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  • 1995-1999  (813)
  • 1975-1979  (1,406)
  • 1920-1924
  • 1997  (813)
  • 1977  (762)
  • 1975  (644)
  • Life and Medical Sciences  (1,942)
  • Electron microscopy  (277)
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  • 1995-1999  (813)
  • 1975-1979  (1,406)
  • 1920-1924
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  • 1
    Electronic Resource
    Electronic Resource
    Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness (1997)
    Springer
    Journal of comparative physiology 181 (1997), S. 425-437 
    Details
    ISSN: 1432-1351
    Keywords: Key words Olfactory receptor cells ; Olfactory bulbectomy ; Olfactory axotomy ; Electrophysiology ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (〉0.1 mmol · l−1) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s003590050126
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  • 2
    Electronic Resource
    Electronic Resource
    Cytodifferentiation of atypical adenomatous hyperplasia and bronchioloalveolar lung carcinoma: immunohistochemical and ultrastructural studies (1997)
    Springer
    Virchows Archiv 431 (1997), S. 415-424 
    Details
    ISSN: 1432-2307
    Keywords: Key words Bronchioloalveolar lung carcinoma ; Atypical adenomatous hyperplasia ; Surfactant apoprotein ; Urine protein 1 ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We used immunohistochemistry and electron microscopy to evaluate the differentiation of cells comprising atypical adenomatous hyperplasia (AAH; n = 26), early bronchioloalveolar lung carcinoma (BAC; n = 11), and overt BAC (n = 16), which are assumed to constitute a continuous spectrum of developmental steps of BAC. Surfactant apoprotein (SAP), a marker for type 2 alveolar cells, was expressed in cells from all the lesions of AAH, early BAC, and overt BAC. However, the proportion of SAP-positive cells decreased and their distribution became more heterogeneous with advancing lesion grade. Urine protein 1, which is identical to the Clara cell-specific 10 kDa protein, was expressed in 70% of overt BAC, whereas only 20% of early BAC showed weak reactivity and none of AAH lesions showed any reactivity at all. Ultrastructurally, type 2 alveolar cell differentiation was predominant among cells from AAH and early BAC. Our results suggest that precursor cells of BAC differentiate predominantly towards type 2 alveolar cells. Cells comprising overt BAC retain this differentiation phenotype, but to a reduced extent. In contrast, concomitantly with progression, cells with Clara cell differentiation emerge and their proportion increases. Such phenotypic changes may reflect metaplasia occurring in tumour cells during the development of BAC.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004280050118
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  • 3
    Electronic Resource
    Electronic Resource
    Adenovirus-induced liver necrosis in a case of AIDS (1997)
    Springer
    Virchows Archiv 431 (1997), S. 469-472 
    Details
    ISSN: 1432-2307
    Keywords: Key words Adenovirus ; Liver pathology ; Electron microscopy ; AIDS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Adenovirus-induced liver necrosis is rare. Before the era of AIDS (acquired immunodeficiency syndrome) this entity was seen predominantly in infants suffering from inborn immunodeficiency syndromes or from iatrogenic immunosuppression because of bone marrow or liver transplantation. Here, we report a case of a 30-year-old woman with AIDS who developed fever and rapidly progressing liver failure. A frozen section from a needle biopsy of the liver allowed a quick diagnosis of viral liver necrosis. The light-microscopic and electron microscopic aspects were typical of adenovirus infection and should be known to the surgical pathologist. The diagnosis was confirmed by immunohistochemistry and DNA hybridization analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004280050125
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  • 4
    Electronic Resource
    Electronic Resource
    Argyrophilic tau-positive twisted and non-twisted tubules in astrocytic processes in brains of Alzheimer-type dementia: an electron microscopical study (1997)
    Springer
    Acta neuropathologica 95 (1997), S. 28-39 
    Details
    ISSN: 1432-0533
    Keywords: Key words Astrocytes ; Alzheimer-type dementia ; Electron microscopy ; Glial fibrillary tangles ; Tau protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract This report concerns pathological astrocytic tubular structures (astrocytic tubules, As-Tbs) that coexist with glial filaments in astrocytic processes in brains with presenile-onset Alzheimer-type dementia. The formation of As-Tbs appears to be related to the duration of disease and the intensity of Alzheimer histopathology. In three cases in which the disease was of extremely long duration, As-Tbs were found in the frontal and temporal neocortices, the temporal pole and the hippocampus using electron microscopy, whereas they were not found in two cases with a long, but not extremely long, illness duration. As-Tbs were almost exclusively found in the highly devastated neuropil, and we could not find them in regions of moderate neuronal degeneration despite intensive inspection. As reported previously, some As-Tbs was seen adjacent to extracellular neurofibrillary tangles (NFTs) and in perivascular astrocytes. Our novel finding is that they can exist independently from these, in the highly devastated neuropil. Two types of As-Tbs were observed, twisted tubules with periodic constrictions at 50- to 80-nm intervals and non-twisted tubules where no constrictions were seen but which had a 15-nm fuzzy outer contour. They were positively stained by anti-human tau antibody, an antibody that does not recognize extracellular NFTs. Thus, it is most likely that As-Tbs are not the sequestration of extracellular NFTs, and that they are of astrocytic origin. Moreover, As-Tbs showed argyrophilia. As-Tbs appear indistinguishable from dystrophic neurites under the light microscope. The present data suggest that they may be more widely distributed in the damaged cerebral neuropil than previously thought.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004010050762
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  • 5
    Electronic Resource
    Electronic Resource
    Scots pine needle injuries at subarctic industrial sites (1997)
    Springer
    Trees 11 (1997), S. 378-387 
    Details
    ISSN: 0931-1890
    Keywords: Key words Pinus sylvestris (L.) ; Electron microscopy ; Heavy metals ; Multi-stress-symptoms ; SO2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract  Injuries to needles of Scots pines (Pinus sylvestris L.) growing in nutrient-poor soils on the Kola Peninsula collected in April 1991 were studied on a gradient of increasing distances (10 – 115 km) from the Monchegorsk nickel smelter, Russia, which emits SO2, Ni and Cu. The condition of the mesophyll cells was quantified from needles of the two latest age classes using a light and an electron microscope. The damage to the ultrastructure consisted of multistress symptoms caused by excess sulphur, heavy metals, frost, acidic precipitation and ozone. Injuries were most commonly manifested in the form of dark, irregularly shaped chloroplasts with protrusions and light thylakoids and plastoglobuli. These symptoms gradually disappeared with increasing distance and decreasing deposition rate. Concentrations of sulphur, copper and nickel decreased towards more distant sites where normal levels of the latter two elements were reached. Sulphur concentrations remained above background throughout the distance gradient. In the closest plots to the smelter area, cell collapse under the stomata and epidermis related to acute SO2 and heavy metal effects was found, whereas further away symptoms were more diverse, pointing towards the effects of ozone, acidic deposition and thereby decreased frost tolerance. The additive multistress symptoms were clearly seen in the area up to 40 km from the smelter where needle Cu concentration was above 110 ppm, Ni concentration above 39 ppm and S concentration above 1343 ppm.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004680050099
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  • 6
    Electronic Resource
    Electronic Resource
    Phenotypic variability of CADASIL and novel morphologic findings (1997)
    Springer
    Acta neuropathologica 94 (1997), S. 247-254 
    Details
    ISSN: 1432-0533
    Keywords: Key words CADASIL ; Electron microscopy ; Granular osmiophilic material ; αB crystallin ; Heat ; shock proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a non-arterio-atherosclerotic, non-amyloidotic arteriopathy affecting preferentially the small arteries and arterioles of the brain. The morphologic hallmark is the presence of a characteristic granular alteration of the arterial media that ultrastructurally corresponds to the accumulation of electron-dense material surrounding the smooth muscle cells. Although the presence of this granular osmiophilic material (GOM) was originally described as limited to brain vessels, identical electron microscopic findings have been demonstrated in the media of peripheral tissue arteries, allowing for a pathologic diagnosis of the disease by a simple skin, muscle or nerve biopsy. We report some atypical features identified in our CADASIL patients that broaden the phenotypic expression of this disease. Firstly, we identified a cortical infarct in an otherwise typical CADASIL patient. Secondly, we observed GOM in skin arteries of a 30-year-old man with hemiplegic migraine, the son of a woman who had died with CADASIL. This confirms that it may be possible to diagnose the disease at a preclinical stage by the ultrastructural evaluation of peripheral tissue biopsy material, particularly for individuals for whom there is a supporting family history. Thirdly, ultrastructural examination of the skin, and subcutaneous and striated muscle of an unrelated and apparently sporadic patient with neuropathologic and neuroradiologic evidence of CADASIL in meningeal and cerebral vessels failed to reveal diagnostic lesions in peripheral arteries. Thus, the possibility of a false-negative pathologic diagnosis in patients with a clinicoradiologic diagnosis of CADASIL, if one relies solely on a peripheral tissue biopsy, does exist. Additionally, we have identified heat shock proteins (Hsp70 and αB crystallin) and ubiquitin in the vascular myocytes of affected arteries. αB crystallin also seemed to be deposited extracellularly, which suggests that GOM also might be immunoreactive for αB crystallin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004010050700
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  • 7
    Electronic Resource
    Electronic Resource
    Density and fine structure of peripheral nerves in various skin lesions of atopic dermatitis (1997)
    Springer
    Archives of dermatological research 289 (1997), S. 125-131 
    Details
    ISSN: 1432-069X
    Keywords: Key words Atopic dermatitis ; Peripheral nerve ;   ; Electron microscopy ; Protein gene product ; Substance P
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The density and fine structure of the peripheral nerve system in various skin lesions of 64 patients with atopic dermatitis (AD) was quantitatively analyzed by immunohistochemical staining with antibodies directed against protein gene product (PGP) and substance P (SP). The density of PGP-positive peripheral nerves was 2.5 × 10 3 μm 2 /Δs (Δs = 0.24 mm 2 selected area) in early acute lesions, 3.8 × 10 3 μm 2 /Δs in subacute lesions, 4.9 × 10 3 μm 2 /Δs in lichenified lesions and 7.1 × 10 3 μm 2 /Δs in prurigo lesions of AD. The density of nerve fibers in subacute, lichenified and prurigo lesions was significantly higher than in uninvolved skin of AD patients (2.0 × 10 3 μm 2 /Δs). Electron microscopically, bulging of axons with many mitochondria and a loss of their surrounding sheath of Schwann cells suggests that the free nerve endings in skin lesions of AD are in an active state of excitation. Many pinocytotic vesicles in the periphery of basal keratinocytes facing nerve endings which contained many neurovesicles suggests reciprocal effects between keratinocytes and nerve endings. The number of SP-positive nerve fibers in AD lesions was far less than one-tenth of the number of PGP-positive nerve fibers.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004030050167
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  • 8
    Electronic Resource
    Electronic Resource
    Effects of brefeldin-A and monensin on organelle distribution and morphology in the preimplantation mouse embryo (1997)
    Springer
    Development genes and evolution 206 (1997), S. 503-514 
    Details
    ISSN: 1432-041X
    Keywords: Key words Preimplantation mouse embryo ; Brefeldin-A ; Monensin ; Golgi ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The intracellular trafficking of integral membrane and secreted proteins is likely to be a key element involved in the morphogenesis and differentiation of the early mammalian embryo. In this study, we used transmission electron microscopy (TEM) to analyse the effects of brefeldin-A (BFA) and monensin, well known inhibitors of vesicular protein trafficking in somatic cells, on the structure of preimplantation mouse embryos. Both BFA and monensin distinctively altered the morphology of Golgi compartments in the blastomeres of treated morulae. BFA-treated morulae lacked recognizable Golgi complexes but possessed heterogeneous organelle clusters consisting of an abundance of smooth tubular and vesicular membrane compartments in addition to mitochondria, endosomes and lysosomes. Treatment of morulae with monensin was associated with swelling of Golgi compartments in addition to altering the morphology of mitochondria, lysosomes and the plasma membrane. BFA, and to a lesser extent monensin, inhibited cytokinesis as evidenced by the detection of binucleate blastomeres. In addition, BFA induced morulae to decompact. These latter effects have not been reported previously for these agents in mammalian somatic cell lines or other vertebrate or invertebrate embryos. These results provide the first demonstration of the structural effects of BFA and monensin on cells of the early mammalian embryo, some of which are consistent with the known actions of these agents on components of the vesicular protein trafficking system in mammalian somatic cells. This information serves as a foundation for the further use of these agents in studies of vesicular protein trafficking as an agent of preimplantation morphogenesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004270050081
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  • 9
    Electronic Resource
    Electronic Resource
    Komibinierte neuronale und endokrine Tumoren der Sellaregion (1997)
    Springer
    Der Pathologe 18 (1997), S. 419-424 
    Details
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Gangliozytom ; Gangliogliom ; Hypophysenadenom ; Immunhistologie ; Ultrastruktur ; Key words Gangliocytoma ; Ganglioglioma ; Pituitary adenoma ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Gangliocytomas or gangliogliomas of the sellar region are very rare tumors. In a great proportion of those cases an adenoma of the anterior pituitary develops from the cell type that is hyperstimulated by the releasing hormone produced from the gangliocytoma. Five GHRH secreting gangliocytomas are reported. Four of these were localized adjacent to a GH secreting adenoma. In one case, no adenoma tissue was found beside the ganglicytoma. As only the adenomas can secrete GH, the adenomas and not the gangliocytomas are directly responsible for acromegaly so that such an adenoma has to be present in cases of acromegaly. A CRH secreting gangliocytoma was combined with an ACTH cell adenoma that had induced Cushing’s disease. A ganglioglioma of the posterior pituitary had led to an inappropriate secretion of Vasopressin. The morphology of the different tumors is presented.
    Notes: Zusammenfassung Intraselläre Gangliozytome/Gangliogliome sind sehr seltene Tumoren, die in einem Großteil der Fälle ein Hypophysenadenom aus dem Zelltyp heraus entstehen lassen, der durch das gebildete Releasinghormon überstimuliert wird. Es wird über 5 GHRH-bildende Gangliozytome berichtet, von denen 4 einem STH-bildenden Hypophysenadenom benachbart waren. Ein Fall enthielt keine Adenomanteile. Diese müssen in Anbetracht der klinisch vorhandenen Akromegalie aber vorhanden sein, da nur das jeweilige Adenom, nicht aber die Gangliozytome für die Überfunktion unmittelbar verantwortlich sind. Ein CRH-bildendes Gangliozytom war mit einem zum Morbus Cushing führenden ACTH-Zelladenom kombiniert. Ein Gangliogliom des Hinterlappens hatte zu einer inadäquaten Vasopressinsekretion Anlaß gegeben. Die Morphologie der Tumoren wird dargelegt.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002920050236
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  • 10
    Electronic Resource
    Electronic Resource
    Ultrastructural subtypes of glutamate-immunoreactive terminals on rat trigeminal motoneurones and their relationships with GABA-immunoreactive terminals (1997)
    Springer
    Experimental brain research 114 (1997), S. 99-116 
    Details
    ISSN: 1432-1106
    Keywords: Key words Synapse ; Axo-axonic synaptic contacts ; Trigeminal motor nucleus ; Immunogold ; Electron microscopy ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Electron-microscopic immunolabelling methods were used to study the relationships between glutamate-immunoreactive and γ-aminobutyric acid (GABA)-immunoreactive synapses on trigeminal motoneurones labelled by the retrograde transport of horseradish peroxidase. Serial sections were cut through the motor nucleus, alternate sections were incubated with antibodies to glutamate and GABA, and the immunopositive nerve terminal profiles were recognized using a quantitative, postembedding immunogold method. Boutons exhibiting high levels of glutamate immunoreactivity and GABA-immunoreactive boutons both formed axo-dendritic and axo-somatic synaptic contacts on labelled motoneurones. Boutons strongly immunopositive for glutamate were not immunopositive for GABA, and vice versa. Strongly glutamate immunoreactive boutons received axo-axonic synaptic contacts but did not form such contacts, while GABA-immunoreactive boutons formed axo-axonic synapses but did not receive them. The presynaptic elements at all axo-axonic synapses on to glutamate-immunoreactive boutons sampled were GABA-immunopositive. These data provide ultrastructural evidence in support of the roles of glutamate and GABA as transmitters at synapses on trigeminal motoneurones, and for presynaptic control of transmission at glutamatergic synapses by GABA acting at receptors at axo-axonic synapses. The vast majority (more than 90%) of strongly glutamate immunoreactive boutons contained spherical synaptic vesicles, in contrast to GABA-immunoreactive boutons, which contained pleomorphic vesicles. Most of the glutamate-immunoreactive boutons (67%) formed asymmetrical synaptic active zones, many of which (47% of total) were associated with subsynaptic dense ”Taxi” bodies (T-terminals), while a smaller population of boutons (21%) formed symmetrical synapses, and a few (11%) made synapses associated with subsynaptic cisternae (C-terminals). The heterogeneity of active zone ultrastructure of boutons identified as being glutamatergic on the basis of their high levels of immunolabelling is discussed in relation to possible differences in co-transmitters released, origins of the synaptic input or post-synaptic receptor subtypes activated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00005627
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  • 11
    Electronic Resource
    Electronic Resource
    Type III Ehlers-Danlos syndrome and pregnancy (1997)
    Springer
    Archives of gynecology and obstetrics 261 (1997), S. 39-43 
    Details
    ISSN: 1432-0711
    Keywords: Key words: Ehlers-Danlos type III ; Pregnancy ; Electron microscopy ; Prenatal diagnosis ; Premature rupture of membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. We report a case of type III Ehlers-Danlos syndrome with a favourable outcome. We review the literature and do not consider that pregnancy in patients with type III Ehlers-Danlos Syndrome represents a high risk situation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004040050196
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  • 12
    Electronic Resource
    Electronic Resource
    Light and electron microscopic investigation of the process of healing of the naevus of Ota by Q-switched alexandrite laser irradiation (1997)
    Springer
    Virchows Archiv 431 (1997), S. 63-71 
    Details
    ISSN: 1432-2307
    Keywords: Key words Naevus of Ota ; Q-switched alexandrite laser ; Light microscopy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Melanocytes in the naevus of Ota were destroyed by irradiation using the Q-switched alexandrite laser. This laser is highly selective and highly absorbed by melanosomes. Other cells and tissue components of the dermis remained almost intact. Melanosomes were vaporized or fragmented to subelectron microscopical size, or degenerated. If the irradiated energy was sufficient, melanocytes vanished and large vacuoles several times the size of dermal melanocytes formed at the sites. If it was too weak, dermal melanocytes were also vaporized, but vacuoles formed within them. Nuclei were no longer discernible. Following irradiation macrophages infiltrated the irradiated areas and scavenged degenerated melanosomes and cellular debris. Thus, discoloration of the skin was markedly reduced. Although a few melanocytes and melanophages remained, pigmentation cleared to a satisfactory level. Melanocytes and keratinocytes were also injured in the epidermis; however, the epidermis recovered completely. No scarring was observed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004280050070
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  • 13
    Electronic Resource
    Electronic Resource
    Neuropeptide Y-immunoreactive intracardiac neurones, granule-containing cells and nerves associated with ganglia and blood vessels in rat and guinea-pig heart (1997)
    Springer
    Cell & tissue research 289 (1997), S. 445-454 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Intracardiac neurones ; Innervation ; Heart ; Neuropeptide Y ; Immunocytochemistry ; Electron microscopy ; Rat (Sprague Dawley) ; Guinea-pig (Dunkin Hartley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Intrinsic neuropeptide Y-containing neurones in rat and guinea-pig hearts were studied at the ultrastructural level by the pre-embedding peroxidase-antiperoxidase immunocytochemical technique. Intracardiac neuronal cell bodies were often weakly or moderately immunostained, and the labelling was usually pronounced in the Golgi complex, multivesicular bodies, some cisterns of granular endoplasmic reticulum and large granular vesicles. Neuropeptide Y-immunoreactive nerve fibres were also observed in association with intracardiac neurones. A subpopulation of neuropeptide Y-immunoreactive granule-containing cells in the rat heart are described for the first time and were very heavily labelled; other granule-containing cells were non-immunoreactive, but were contacted by neuropeptide Y-containing nerves. Preterminal regions of nerve fibres that were located in nerve bundles were only weakly neuropeptide Y-immunoreactive, in contrast to the heavy labelling observed in varicosities that contained many synaptic vesicles. Many neuropeptide Y-immunoreactive nerve fibres were associated with the coronary vasculature and were particularly prominent in the walls of small arteries and arterioles where labelled nerve varicosities were present close to the smooth muscle cells. Immunoreactive nerves were also seen in the myocardium, usually near to capillaries. In axonal varicosities, the central core of large granular vesicles was immunolabelled, and electron-dense immunoreactive material outlined the membranes of small and large clear vesicles. The significance of neuropeptide Y-immunoreactive intracardiac neurones and granule-containing cells and the origin of associated labelled nerve fibres in the heart are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050890
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  • 14
    Electronic Resource
    Electronic Resource
    Immuno-electron microscopy reveals that the excitotoxin quinolinate is associated with the plasma membrane in human peripheral blood monocytes/macrophages (1997)
    Springer
    Cell & tissue research 290 (1997), S. 633-639 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Quinolinic acid ; Interferon-γ ; Kynurenine ; Electron microscopy ; Immunocytochemistry ; Excitotoxicity ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract.  Quinolinate (QUIN), a tryptophan-derived excitotoxin, was localized ultrastructurally in human peripheral blood monocytes/macrophages (MØ) by immuno-electron microscopy. A combined carbodiimide/glutaraldehyde/paraformaldehyde-based fixation procedure was developed for optimal retention of QUIN in the cell as well as minimal loss of ultrastructure; a silver-enhanced colloidal gold detection system was used for electron-microscopic analysis. Gold particles representing QUIN immunoreactivity were associated with the inner side of the plasma membrane in normal MØ. The number of gold particles increased significantly when QUIN levels were elevated by treatment with its precursor kynurenine, but location of the gold particles remained essentially the same under this condition. Treatment with interferon-γ increased the number of Golgi bodies, vacuoles and pseudopodia, reflecting the activated state of the cell. Significantly increased numbers of gold particles representing QUIN were detectable in approximately the same location as in the case of kynurenine treatment. Combined treatment with kynurenine and interferon-γ maximally increased the number of gold particles at the periphery of the cell. The pseudopodia were intensely stained with gold particles, while they were not detectable in the inner part of the cytoplasm or in any other organelle even under this activated condition. The significance of the specific location of QUIN revealed in the present study and its relation to the release and subsequent actions of QUIN are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050969
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  • 15
    Electronic Resource
    Electronic Resource
    Synaptic exocytosis of dense-core vesicles in blue crab (Callinectes sapidus) stomach muscles (1997)
    Springer
    Cell & tissue research 289 (1997), S. 517-526 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Neuromuscular junction ; Synaptic vesicles ; Vesicle fusion ; Electron microscopy ; Callinectes sapidus (Crustacea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Neuromuscular terminals of a single motoneuron to four muscles (CPV7a, GM5a, CV2, and CV3) in the stomach of the blue crab Callinectes sapidus showed structural evidence for the exocytotic release of dense-core vesicles exclusively at synapses. The primary evidence was the appearance of dense cores in the synaptic cleft, accompanied by indentations of the presynaptic or postsynaptic membrane. In their simplest form, these consisted of an omega-shaped figure of the presynaptic membrane enclosing one dense core, denoting release of a single dense-core vesicle. A larger indentation of the presynaptic membrane enclosing several dense cores denoted multiple release. A more complex form of multiple release was where the presynaptic membrane was normal, but the postsynaptic membrane elaborated into a sac projecting into the granular sarcoplasm and filled with dense cores. The postsynaptic sac in some instances was compressed into a thin, fingerlike extension, which lacked dense cores and, at its distal end, separated into small cisternae, suggesting a mechanism for membrane recycling. Profiles depicting single and multiple releases of dense-core vesicles were found more frequently at neuromuscular terminals that release relatively large amounts of transmitter with a single stimulus, such as CV2 and CV3, compared to those releasing smaller amounts, such as CPV7a and GM5a. The disparity in release sites among the four muscles of this single motor unit and the fact that many of the multiple-release figures were closely adjacent to the active zones for transmitter release suggest a possible modulatory role for dense-core vesicles in synaptic transmission. Such modulation may be long lasting, as implied by the postsynaptic sacs, which may permit prolonged release of the contents of their dense cores into the synaptic cleft. This is in keeping with the functional role of these stomach muscles, which is to be continuously active for long periods of time.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050897
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  • 16
    Electronic Resource
    Electronic Resource
    Immunoreactive excitatory amino acids in the parietal eye of lizards, a comparison with the pineal organ and retina (1997)
    Springer
    Cell & tissue research 287 (1997), S. 275-283 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Parietal eye ; Pineal organ ; Retina ; Glutamate ; Aspartate ; Immunocytochemistry ; Electron microscopy ; Lacerta muralis ; Lacerta agilis ; Lacerta viridis (Lacertilia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The fine structure of the organ and the localization of the excitatory amino acids glutamate and aspartate were studied in the parietal eye of lizards by postembedding immunoelectron microscopy. The parietal eye contains cone photoreceptor cells, secondary neurons, and ependymal and lens cells. The photoreceptors form long inner and outer segments, some of them being paired as ”twin-photoreceptors” by zonulae adherentes. Perikarya of neurons bear sensory cilia (containing 9×2+0 pairs of tubules) extending into the intercellular space. No neurohormonal terminals are present in the parietal eye. A higher immunoreactivity to glutamate than to aspartate is found in the photoreceptors and in the secondary neurons of the parietal eye. Glutamate immunogold labeling is more intense in the axonal processes of photoreceptors and neurons and in most of the nerve fibers of the parietal nerve running to the brain stem. Weak aspartate and glutamate immunoreactivity can be detected in the ependymal and lens cells. A similar distribution of immunoreactive amino acids is found in the photoreceptors, secondary neurons, and ependymal glial elements of the pineal organ, and retina of the lateral eye of the same animals. Immunoreactive glutamate accumulates in the axons of photoreceptors and secondary neurons of the parietal eye suggesting that this excitatory amino acid acts as a synaptic mediator in the neural efferentation of the organ. Thus, the efferent light-conducting pathway of the parietal organ is similar to that of the pineal organ and lateral eye retina. As the Mullerian cells of the retina, the ependymal and lens cells of the parietal eye and the ependymal-glial cells of the pineal organ may play a role in the metabolism and/or elimination of excitatory amino acids released by photoreceptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050752
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  • 17
    Electronic Resource
    Electronic Resource
    Expression of basic fibroblast growth factor-like immunoreactivity in the nuclei of regenerating hepatocytes (1997)
    Springer
    Cell & tissue research 288 (1997), S. 517-527 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Liver ; Subcellular fractions ; Immunohistochemistry ; Electron microscopy ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. This study, utilizing rats subjected to two-thirds partial hepatectomy or sham operation, was designed (1) to investigate the content of basic fibroblast growth factor (bFGF) in the subcellular fractions of regenerating and sham-operated rat livers by immunoblot experiments and enzyme-linked immunosorbent assay (ELISA), (2) to show that bFGF immunoreactivity and proliferating cell nuclear antigen (PCNA) immunoreactivity are markers for hepatocellular mitosis before and after partial hepatectomy, and (3) to observe the location and fine structure of the bFGF immunoreaction within the regenerating liver with special attention to bFGF immunoreactivity in the nuclei of regenerating hepatocytes. Immunoblot experiments and ELISA showed a transient increase in high-molecular-weight forms of bFGF in the nuclear subcellular fraction of regenerating liver 48 h after partial hepatectomy. By light microscopy, bFGF and PCNA immunoreactivities were detected in the nuclei of regenerating hepatocytes. Electron microscopy demonstrated bFGF-like immunoreactivity mainly in the nuclear euchromatin and rarely in the heterochromatin or nucleoli of regenerating hepatocytes. The transient increase in high-molecular-weight forms of bFGF in the nuclear euchromatin of regenerating hepatocytes, together with the concomitant expression of PCNA in the regenerating liver, suggests an important role of the high-molecular-weight forms of bFGF in hepatocyte proliferation and/or mitosis, although authentic bFGF with a molecular form of 18 kDa is not considered to be involved in hepatic regeneration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050837
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  • 18
    Electronic Resource
    Electronic Resource
    Osmoregulatory mutants that affect the function of the contractile vacuole inChlamydomonas reinhardtii (1997)
    Springer
    Protoplasma 200 (1997), S. 99-111 
    Details
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; Contractile vacuole ; Electron microscopy ; Videomicroscopy ; Osmoregulation ; Osmoregulatory mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01280738
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  • 19
    Electronic Resource
    Electronic Resource
    Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii (1997)
    Springer
    Protoplasma 198 (1997), S. 73-84 
    Details
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; Contractile vacuole ; Osmoregulation ; Videomicroscopy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01282133
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  • 20
    Electronic Resource
    Electronic Resource
    Selective UV-induced damage to short-wavelength receptor cells in the butterflyPapilio xuthus (1997)
    Springer
    Protoplasma 200 (1997), S. 112-115 
    Details
    ISSN: 1615-6102
    Keywords: Electron microscopy ; UV-induced cell damage ; Photoreceptor ; Eye ; Lepidoptera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A technique that allows the specific inactivation of short-wavelength-sensitive photoreceptive cells has been needed for a long time. Such a technique could be useful in studies on the role(s) of UV-receptors in circadian rhythmicity, recognition of floral patterns, homing behaviour, and mate selection in arthropods. We provide ultrastructural evidence that short-wavelength receptor cells can be selectively damaged without affecting other spectral-cell types. Since the method does not require the killing of the experimental animal, the latter can be used in behavioural or other follow-up tests.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01280739
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  • 21
    Electronic Resource
    Electronic Resource
    Electron microscopic and immunomorphological investigations on the mucosa of the human paranasal sinuses (1997)
    Springer
    European archives of oto-rhino-laryngology and head & neck 254 (1997), S. 230-235 
    Details
    ISSN: 1434-4726
    Keywords: Paranasal sinus mucosa ; Collagen type VII ; Subepithelial connective tissue ; Electron microscopy ; Immunofluorescence microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The morphology of the mucosa from the human paranasal sinuses was investigated by electron microscopy. A total of 27 specimens was taken from 11 patients following midfacial fractures. All tissue samples were biopsied during surgery after informed consent had been given. In accordance with light microscopic investigations, the mucosa represented a highly prismatic epithelium consisting of kinocilia-carrying and mucus-producing (goblet) cells. Other cell types, such as those occurring in the respiratory epithelium of other areas, could not be demonstrated. Electron microscopic and immunomorphological investigations revealed collagen type VII beneath the lamina densa of the basal lamina. According to findings obtained to date, this collagen type accompanies only a multilayered epithelium. Another peculiarity was the small number of basophils and eosinophils. Pronounced acute reactions of the mucosa in this area cannot be expected, which is in contrast to that of the nasal mucosa.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00874094
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  • 22
    Electronic Resource
    Electronic Resource
    Ultrastructural findings of the macula utriculi in a case of a petrous apex cholesteatoma: a comparison with findings in a patient with an acoustic neuroma (1997)
    Springer
    European archives of oto-rhino-laryngology and head & neck 254 (1997), S. 255-258 
    Details
    ISSN: 1434-4726
    Keywords: Electron microscopy ; Inner ear ; Utricle ; Cholesteatoma ; Acoustic neuroma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The morphological characteristics of the vestibular sensory cells of the macula utriculi obtained during surgery in a patient with a petrous apex cholesteatoma were examined using scanning and transmission electron microscopy. Findings were compared to cells studied in a patient with acoustic neuroma. Scanning electron microscopy showed that compared to the apparently normal cells in the acoustic neuroma case, most sensory cells in the cholesteatoma case had large cuticular plates, irregular locations of cilia and no clear polarizations. Supporting cells showed profuse short microvilli on the whole surface. With transmission electron photomicrographs, type I hair cells were not seen and certain morphological changes were observed in type-1I-like cells and supporting cells. We presume that the degenerative changes in the vestibular epithelia were due to circulatory disturbances and/or direct pressure applied to the vestibular nerve at the internal auditory canal, with subsequent involvement of the macula utriculi.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00874100
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  • 23
    Electronic Resource
    Electronic Resource
    Electron microscopic localization of nitric oxide I synthase in the organ of Corti of the guinea pig (1997)
    Springer
    European archives of oto-rhino-laryngology and head & neck 254 (1997), S. 396-400 
    Details
    ISSN: 1434-4726
    Keywords: Guinea pig hair cells ; Electron microscopy ; Immunocytochemistry ; Nitric oxide synthase ; Sound transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Nitric oxide synthase (NOS) activity has been detected previously in the mammalian cochlea at a light microscopic level. Here we present results of electron microscopic analysis for post-embedding immunoreactivity of neural-type NOS I in the cochlea of the guinea pig. Strong enzyme immunoreactivity was identified in the cytoplasm of inner and outer hair cells. Gold-labeled NOS I antibodies were mainly located in electron-dense areas of the cytoplasm, whereas electron-lucent regions of the receptor cells were nearly free from any immunoreactivity. In both types of hair cells anti-NOS I antibodies were also visible in the cuticular plates, hair bundles and nuclei. Further ultrastructural analysis revealed that the submembranous cisternae of the outer hair cells were nearly free from any reaction product, demonstrating that the whole cytoplasm of this hair cell was not immunoreactive. Other NOS I immunoreactivity was identified in the cuticular plates of the inner and outer pillar cells and in the cytoskeletal elements located in the apical parts of Deiter cells, forming the lamina reticularis or in cytoskeletal-containing regions in basal Deiter cells. Anti-NOS antibodies were visible in the nuclei of various cell types. Our findings suggest that nitric oxide produced by NO I synthase in the organ of Corti may act as a modulator of hair cell physiology during the processes of signal transduction with frequence selectivity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01642558
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  • 24
    Electronic Resource
    Electronic Resource
    Immunohistochemical and electron microscopical characterization of stromal cells in nasopharyngeal angiofibromas (1997)
    Springer
    European archives of oto-rhino-laryngology and head & neck 254 (1997), S. 196-199 
    Details
    ISSN: 1434-4726
    Keywords: Nasopharyngeal angiofibroma ; Ultrastructure ; Myofibroblast ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Twenty-eight cases of nasopharyngeal angiofibroma were studied immunohistochemically for cytoskeletal phenotyping of stromal cells. Electron microscopy was also used to study the ultrastructure of five of the tumors. All typical stromal cells showed intensive immunostaining for vimentin, but were negative for smooth muscle actin and desmin. Ultrastructurally, most of these cells appeared to be exclusively fibroblasts. However, in some areas stromal cells were seen that morphologically resembled myofibroblasts by their shapes and arrangement, and were characterized by the coexpression of vimentin and smooth muscle actin. Electron microscopy confirmed their myofibroblastic nature. The present study showed that the typical stromal cells in nasopharyngeal angiofibromas were fibroblasts and not myofibroblasts. In these tumors myofibroblasts occurred only focally, in connection with fibrotic areas and exclusively as a vimentin+/actin+ cytoskeletal phenotype. This indicates that myofibroblasts are not primary stromal tumor cells in nasopharyngeal angiofibromas, but occur due to regressive changes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00879273
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  • 25
    Electronic Resource
    Electronic Resource
    Morphological and cytochemical changes in human mast cells during culture (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 25-30 
    Details
    ISSN: 1860-1499
    Keywords: Mast cell ; Culture ; Electron microscopy ; Carbohydrate cytochemistry ; Peroxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Mast cells were cultured from human umbilical cord blood CD34-positive cells in the presence of interleukin (IL)-6 and stem cell factor (SCF). Electron microscopically, a particulate or beaded structure, the prominent structure of tryptase-positive mast cell (T mast cell) granules, was frequently observed in 54-day cultures. Straight or coiled lamellae, often observed in the granules of tryptase-and chymase-positive mast cells (TC mast cells), were observed in the granules in 75-day cultures. Cytochemically, the peroxidase activity of the granules was positive but decreased during the term of culture. Glycogen particles were abundant in 54-day cultures, and scarce in 75-day cultures. These findings suggest that the mast cells matured and changed in phenotype during the culture's term.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01458348
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  • 26
    Electronic Resource
    Electronic Resource
    An ultrastructural study of pemphigoid vegetans, characterizing the possible mechanism of vegetation (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 48-54 
    Details
    ISSN: 1860-1499
    Keywords: Pemphigoid vegetans ; Lamina densa ; Mast cell ; Vegetation ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Ultrastructural changes in the skin lesions of pemphigoid vegetans were observed by transmission electron microscopy. Two significant changes were recognized at the vegetating site: (1) The basal lamina was sometimes interrupted and detached from the basal cells, protruding into the dermis to form folds. In the basal cells, hemidesmosomes were not decreased in number but tonofilament bundles were fewer than normal. Occasionally, basal cell processes extended beyond the basal lamina through its gap. These features resembled the epidermal growth factor-induced morphological changes of cultured skin. It was suspected that the abnormal attachment of the basal lamina to the basal cell was associated with keratinocyte proliferation. (2) Mast cells were encountered in the epidermis abutting on the basal lamina. It is uncommon to find mast cells in the epidermis. Mast cells in the epidermis were reported at the erythematous regions which were adjacent to the bullae of bullous pemphigoid, and were thought to have an important role in forming blisters. It was suggested that early changes in blister formation occur at the vegetating site.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01458352
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  • 27
    Electronic Resource
    Electronic Resource
    Morphological changes of c-Fos-like immunoreactivity in rat cerebral cortex after cerebral ischemia and reperfusion with special reference to vasoactive intestinal polypeptide (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 111-118 
    Details
    ISSN: 1860-1499
    Keywords: c-Fos ; VIP ; Electron microscopy ; Immunocytochemistry ; Cerebral ischemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We investigated morphological changes in neurons with c-Fos-like immunoreactivity (c-Fos-LI) after cerebral ischemia by light and electron microscopic immunocytochemistry. Strong c-Fos-LI was observed in layers II–VI of the cerebral cortex with an especially abundant distribution in the nuclei of layers II, IV, and VI ipsilateral to the lesioned side. Reperfusion after ischemia had a greater effect on the expression of c-Fos-LI than did permanent ischemia. Vasoactive intestinal peptide (VIP)-positive neurons were seen scattered in layers II–V of the cerebral cortex. Some VIP-positive neurons showed c-Fos-LI after ischemia. Electron microscopy revealed c-Fos-LI in euchromatin in the nuclei of c-Fos-positive cells. Dilatation of the cisternae of the rough endoplasmic reticulum and the presence of numerous secondary lysosomes were found in neurons on the lesioned side after 12h of reperfusion. Some VIP-containing neurons revealed c-Fos-LI with reperfusion after ischemia by a double immunostaining method on the same tissue section. These findings suggest that ischemia potentiates c-fos expression in VIP- or other transmitter- or modulator-containing neurons, thereby protecting from neuronal cell death.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545311
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  • 28
    Electronic Resource
    Electronic Resource
    Morphological changes ofHelicobacter pylori after antibacterial therapy: an electron microscope study (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 131-137 
    Details
    ISSN: 1860-1499
    Keywords: Helicobacter pylori ; Electron microscopy ; Three-dimensional reconstruction ; Eradication ; 13C-urease breath test ; Coccoid form
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To investigate the effect of morphological change ofHelicobacter pylori on gastric mucosal inflammation,H. pylori was examined by means of an electron microscope and computer-assisted three-dimensional reconstruction before and after antibacterial therapy. The results showed that the bacterium was usually present in different forms and sizes. Most of these variations are helicobacter shaped, 3–5 μm long (43%), or campylobacter shaped, 1–3 μm long (38%); fewer are round or coccoid (19%), and only 0.5–1 μm long. Active inflammation and an abnormal amount of mucus in the epithelial cells are the main changes seen in gastric mucosa withH. pylori infection. In some cases, 6 weeks after antibacterial therapy,H. pylori could still be observed by toludine blue, with slight positive results withH. pylori-antibody-labeled immunostaining. In contrast to the bacteria before antibacterial therapy, in mostH. pylori-positive cases usually only a minute number of bacteria were seen in coccoid form. These surviving coccoid-form bacteria showed thickened walls, and the surrounding periplasmic space was reduced or nonexistent. The epithelial cells were almost normal and13C-text negative. Although neutrophil infiltration was seldom seen, there were still some surviving bulblike cells with excess amounts of mucus in the gastric pits. It is suggested that the coccoid form ofH. pylori may be a static bacterium without urease production. For therapeutic decisions, it may be more important to identify whether there is an active inflammation in the gastric mucosa than to detectH. pylori.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545314
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  • 29
    Electronic Resource
    Electronic Resource
    Aneurysmal (angiomatoid) fibrous histiocytoma of the skin: an unusual variant of dermatofibroma (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 163-169 
    Details
    ISSN: 1860-1499
    Keywords: Dermatofibroma ; Aneurysmal ; (angiomatoid) fibrous histiocytoma ; Immunohistochemistry ; Electron microscopy ; Skin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We present a rare case of aneurysmal (angiomatoid) fibrous histiocytoma (AAFH) of the skin on the back of a 40-year-old Japanese man. Histologically, the tumor was characterized by massive proliferation of fibroblastic and histiocytic cells, prominent aggregation of hemosiderin pigment, and the presence of blood-filled tissue spaces devoid of an endothelial lining within a capillaryrich stroma. Immunohistochemically the tumor cells were immunoreactive for vimentin and for Factor XIIIa or Mac387. Ultrastructural study revealed that the tumor was composed mainly of fibroblast-like cells intermingled with histiocyte-like cells and intermediate cells with combined features of the two types of cells. These findings support the fibrohistiocytic origin of aneurysmal (angiomatoid) fibrous histiocytoma. In addition, ultrastructural examination seems quite useful to differentiate from other cutaneous neoplasms with architectural and cytological similarities to this tumor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545318
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  • 30
    Electronic Resource
    Electronic Resource
    Osteoclast-like giant cell tumor of the pancreas: an immunohistochemical and ultrastructural study (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 216-220 
    Details
    ISSN: 1860-1499
    Keywords: Osteoclast-like giant cell tumor ; Pancreas ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report a rare case of osteoclast-like giant cell tumor of the pancreas in a 70-year-old Japanese woman. The tumor was composed of a proliferation of ovoid to spindle-shaped mononuclear cells admixed with osteoclast-like giant cells. The tumor cells were immunore-active for vimentin, ±1-antitrypsin, and CD68. In ultrastructural examination, the giant cells resembled osteoclasts, and the mononuclear stromal cells had fibroblastic and histiocytic features. No elements of epithelial differentiation were found in this tumor. These findings suggest that this tumor had a derivation similar to giant cell tumor of bone.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545773
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  • 31
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    Electronic Resource
    An electron microscopic and immunohistochemical study of Merkel cell carcinoma (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 209-215 
    Details
    ISSN: 1860-1499
    Keywords: Merkel cell carcinoma ; Dense core granule ; Chromogranin A ; Electron microscopy ; Immunohistochemistry study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Two cases of Merkel cell carcinoma (MCC) were examined by electron microscopy and immunohistochemistry. Histologically, tumor cells, which extended from the dermis into the subcutis, showed anastomosing bands with partial trabecular pattern. The ultrastructural study showed tumor cells in case 1 with numerous neurosecretory granules. The number of granules in case 2, however, was smaller compared with that in case 1. Perinuclear bundles of filaments were present in case 2, but few bundles were observed in case 1. By immunohistochemistry, cytokeratin (CK)-8, -18, -19, and -20 and epithelial membrane antigen were stained positively within tumor cells in both cases. It was interesting that staining patterns of chromogranin A and of neuron-specific enolase were different in the two cases. These data indicated that CK-20 is a useful marker for diagnosing MCC and that ultrastructural and immunohistochemical differences in both cases were the result of phenotypic variation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545772
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  • 32
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    Electronic Resource
    Cervical cord ependymoma with numerous microrosettes (1997)
    Springer
    Brain tumor pathology 14 (1997), S. 145-151 
    Details
    ISSN: 1861-387X
    Keywords: Microrosette ependymoma ; Spinal cord ependymoma ; Trabecular architecture ; Immunohis-tochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract “Microrosette ependymoma,” which is ependymoma with numerous microrosettes throughout the tumor, has rarely been reported. We describe an autopsy case of cervical cord ependymoma with two unusual features: the presence of numerous microrosettes and the formation of trabecular architecture. The tumor originated in the C2 segment of a man aged 23 years and gradually expanded over the following 15 years and 10 months until the entire cervical cord was involved. Beside the low grade of malignancy, the tumor cells exhibited a strong tendency to form microrosettes and trabecular architecture, which formed many perivascular pseudorosettes. The microrosettes mostly consisted of only two or a few more cells, in the absence of large rosettes. Thus the constituent cells were those forming perivascular pseudorosettes. Electron microscopy and immunohistochemistry characterized the ependymal properties of the microrosettes, whose lumina frequently contained fibril bundles similar to those of the Reissner's fiber fibrils, in addition to cilia and microvilli. The pathogenesis of the occurrence of numerous microrosettes is unknown; however, a defect in the mechanism of regulation of rosette formation and enlargement is the most likely explanation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02478884
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  • 33
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    Electronic Resource
    Gastric carcinoid tumors in a brother-sister occurrence, with special reference to ultrastructural features of endocrine granules (1997)
    Springer
    Medical molecular morphology 30 (1997), S. 228-234 
    Details
    ISSN: 1860-1499
    Keywords: Gastric carcinoid tumors ; Familial occurrence ; Electron microscopy ; Endocrine granules
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Our report concerns gastric carcinoid tumors in a man and his younger sister who had no endocrine symptoms. The tumors in both cases were multiple, located in the corpus, and showed no significant differences in light microscopic and immunohistochemical examination. In electron microscopic observation, the endocrine granules of tumor cells were roughly classified into four types: granules with fine granular content with low electron density, irregularly striped granules with medium electron density, concentrically laminated or regularly striped granules, or smaller and almost uniformly high electron-dense granules in which it was difficult to recognize the internal structure. From the sequential morphological changes of the granules, we have considered that each type of granule reflects various stages of granulogenesis, although the possibility that they are distinct and contain different hormones or chemical mediators is not absolutely ruled out.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01545775
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  • 34
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    Electronic Resource
    Ultrastructure of microvessels and tumor cell processes on central neurocytoma (1997)
    Springer
    Brain tumor pathology 14 (1997), S. 79-83 
    Details
    ISSN: 1861-387X
    Keywords: Neurocytoma ; Intraventricular tumor ; Electron microscopy ; Blood vessel ; Tumor cell process
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A case of central neurocytoma that was confirmed with ultrastructural and immunohistochemical studies has been reported. Ultrastructurally, thin cytoplasmic processes of tumor cells showed differentiation into neuronal cells containing parallel bundles of microtubules and abortive synapses with dense-core vesicles and/or clear vesicles. It was frequently found that the clusters of tumor cell processes were close to or around the microvessels. Microvessels were composed of endothelial cells without fenestrations and had tight junctions in the endothelial clefts. Neurosecretory granules in thin cell processes appeared close to microvessels and may have been secreted around microvessels.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02478874
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    Detection of a proliferation specific gene during development of the osteoblast phenotype by mRNA differential display (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 106-116 
    Details
    ISSN: 0730-2312
    Keywords: osteoblasts ; proliferation ; growth control ; differential display ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fetal rat calvarial-derived osteoblasts in vitro (ROB) reinitiate a developmental program from growth to differentiation concomitant with production of a bone tissue-like organized extracellular matrix. To identify novel genes which may mediate this sequence, we isolated total RNA from three stages of the cellular differentiation process (proliferation, extracellular matrix maturation, and mineralization), for screening gene expression by the differential mRNA display technique. Of 15 differentially displayed bands that were analyzed by Northern blot analysis, one prominent 310 nucleotide band was confirmed to be proliferation-stage specific. Northern blot analysis showed a 600-650 nt transcript which was highly expressed in proliferating cells and decreased to trace levels after confluency and throughout the differentiation process. We have designated this transcript PROM-1 (for proliferating cell marker). A full length PROM-1 cDNA of 607 bp was obtained by 5′ RACE. A short open reading frame encoded a putative 37 amino acid peptide with no significant similarity to known sequences. Expression of PROM-1 in the ROS 17/2.8 osteosarcoma cell line was several fold greater than in normal diploid cells and was not downregulated when ROS 17/2.8 cells reached confluency. The relationship of PROM-1 expression to cell growth was also observed in diploid fetal rat lung fibroblasts. Hydroxyurea treatment of proliferating osteoblasts blocked PROM-1 expression; however, its expression was not cell cycle regulated. Upregulation of PROM-1 in response to TGF-β paralleled the stimulatory effects on growth as quantitated by histone gene expression. In conclusion, PROM-1 represents a small cytoplasmic polyA containing RNA whose expression is restricted to the exponential growth period of normal diploid cells; the gene appears to be deregulated in tumor derived cell lines. J. Cell. Biochem. 64:106-116. © 1997 Wiley-Liss, Inc.
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    Age-related changes in bone formation, osteoblastic cell proliferation, and differentiation during postnatal osteogenesis in human calvaria (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 128-139 
    Details
    ISSN: 0730-2312
    Keywords: osteoblasts ; calvaria ; bone formation ; proliferation ; differentiation ; osteogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (3H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)2, vitamin D3, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and β-glycerophosphate, providing a new model to study human osteogenesis in vitro. J. Cell. Biochem. 64:128-139. © 1997 Wiley-Liss, Inc.
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    The interleukin-1β converting enzyme family of cysteine proteases (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 2-10 
    Details
    ISSN: 0730-2312
    Keywords: ICE ; cysteine proteases ; inflammation ; apoptosis ; Ced3 ; secretion ; cell activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interleukin-1β converting enzyme (ICE) is the first enzyme of a new family of cysteine endoproteinases to be isolated and characterized. An overview of the structure and activity of ICE is outlined together with highlights of salient features common to members of each of the family members. J. Cell. Biochem. 64:2-10. © 1997 Wiley-Liss, Inc.
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    Characterization of mice deficient in interleukin-1β converting enzyme (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 27-32 
    Details
    ISSN: 0730-2312
    Keywords: interleukin-1β converting enzyme ; gene targeting ; apoptosis ; IL-1β ; IL-1α ; inflammation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27-32. © 1997 Wiley-Liss, Inc.
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    In vitro and in vivo studies of ICE inhibitors (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 19-26 
    Details
    ISSN: 0730-2312
    Keywords: ICE ; protease ; interleukin-1 ; cytokine ; programmed cell death ; apoptosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interleukin-1β-converting enzyme (ICE) is a cysteine protease responsible for proteolytic activation of the biologically inactive interleukin-1β precursor to the proinflammatory cytokine. ICE and homologous proteases also appear to mediate intracellular protein degradation during programmed cell death. Inhibition of ICE is a new antiinflammatory strategy being explored by the design of both reversible inhibitors and irreversible inactivators of the enzyme. Such compounds are capable of blocking release of interleukin-1β from human monocytes. ICE inhibitors that cross react against multiple ICE homologs can also block apoptosis in diverse cell types. ICE inhibitors impart protection in vivo from endotoxin-induced sepsis and collagen-induced polyarthritis in rodent models. Further optimization of the current generation of peptidyl ICE inhibitors will be required to produce agents suitable for administration in chronic inflammatory and neurodegenerative diseases. J. Cell. Biochem. 64:19-26. © Wiley-Liss, Inc.
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    Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 278-294 
    Details
    ISSN: 0730-2312
    Keywords: osteoblast ; differentiation ; replication ; osteoprogenitor ; bone marrow ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278-294. © 1997 Wiley-Liss, Inc.
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    Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 328-341 
    Details
    ISSN: 0730-2312
    Keywords: Lysyl oxidase ; type I collagen ; myofibroblast ; fibrosis ; mRNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (α-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type III collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5′-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to -192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter. J. Cell. Biochem. 64:328-341.
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    Localization of group IIc low molecular weight phospholipase A2 mRNA to meiotic cells in the mouse (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 369-375 
    Details
    ISSN: 0730-2312
    Keywords: testis ; phospholipase A2 ; cDNA sequence ; in situ hybridization ; mouse ; pla2g2c ; spermatocytes ; meiosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We use in situ hybridization to demonstrate that the testicular expression of a novel, mouse, low molecular weight phospholipase A2 (PLA2 Group IIc) mRNA is specific to cells undergoing meiosis. A complete cDNA (1421 bp) encoding the mouse Pla2g2c gene was generated with reverse transcription-PCR (RT-PCR) and 5′ and 3′ RACE (rapid amplification of cDNA ends) RT-PCR, and its nucleotide sequence was determined. Northern blots of RNA from different tissues revealed a single 1.6 kb transcript only in testis. In situ hybridization indicated that this mouse gene is transcribed mainly in pachytene spermatocytes, secondary spermatocytes, and round spermatids. Expression of the gene is seen in all stages of the seminiferous epithelium, especially in stages VI-VII. J. Cell. Biochem. 64:369-375. © 1997 Wiley-Liss, Inc.
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    Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 376-381 
    Details
    ISSN: 0730-2312
    Keywords: endothelin-1 ; phospholipase D ; arachidonic acid ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a previous study, we have shown that endothelin-1 (ET-1) activates phospholipase D independently from protein kinase C in osteoblast-like MC3T3-E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET-1-induced arachidonic acid release and prostaglandin E2 (PGE2) synthesis in osteoblast-like MC3T3-E1 cells. ET-1 stimulated arachidonic acid release dose-dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET-1-induced arachidonic acid release in a dose-dependent manner as well as the ET-1-induced diacylglycerol formation. 1,6-bis-(cyclohexyloxyminocarbonylamino)-hexane (RHC-80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET-1-induced arachidonic acid release. The pretreatment with propranolol and RHC-80267 also inhibited the ET-1-induced PGE2 synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET-1 in osteoblast-like cells. J. Cell. Biochem. 64:376-381. © 1997 Wiley-Liss, Inc.
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    Organization of the receptor-mediated phosphoinositide cycle: Relationship between receptor occupancy and accession of phosphatidylinositol (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 382-389 
    Details
    ISSN: 0730-2312
    Keywords: tissue culture ; vasopressin ; signal transduction ; compartmentation ; internalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have previously reported the existence of separate hormone-responsive and -unresponsive pools of inositol phospholipids in WRK-1 cells. In order to further explore this concept, we have performed experiments to examine the relationship between the plasma membrane receptor and the pool of phosphatidylinositol (Ptdlns) that is metabolized in response to hormonal stimulation. The results support the following conclusions. 1) The amount of Ptdlns metabolized in WRK-1 cells in response to vasopressin is proportional to the number of receptors occupied; neither prolonged activation with nor readdition of a submaximal concentration of vasopressin induced the same degree of Ptdlns metabolism as a maximal concentration of vasopressin. 2) Dissociation of cytoskeletal structures by incubation with cytochalasin D did not alter the amount of Ptdlns accessed during hormonal stimulation. 3) Accession of Ptdlns from internal membranes does not depend on internalization and recycling of the receptor; cells incubated in potassium-free medium failed to internalize receptor-ligand complexes, yet they accessed the same amount of Ptdlns in response to vasopressin as did control cells. 4) Golgi-mediated phosphatidylinositol transport is not involved in hormone-stimulated phosphoinositide turnover, since brefeldin A, which interferes with Golgi-mediated transport processes, had no effect on the amount of Ptdlns accessed during vasopressin stimulation. 5) Phosphoinositide breakdown and compensatory resynthesis is not a closed process; newly synthesized Ptdlns is not preferentially localized to a hormone-responsive pool but is generally redistributed between responsive and unresponsive pools. J. Cell. Biochem. 64:382-389. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Production of sulfated proteoglycans by human breast cancer cell lines: Binding to fibroblast growth factor-2 (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 605-617 
    Details
    ISSN: 0730-2312
    Keywords: breast cancer ; proteoglycans ; heparan sulfate ; chondroitin sulfate ; sulfation ; fibroblast growth factor-2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [35S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (Kav 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (Kav 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind 125I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties. J. Cell. Biochem. 64:605-617. © 1997 Wiley-Liss, Inc.
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    Association of protein kinase CK2 with nuclear matrix: Influence of method of preparation of nuclear matrix (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 499-504 
    Details
    ISSN: 0730-2312
    Keywords: protein kinase CK2 ; nuclear matrix ; cytoskeleton ; chromatin ; intermediate filaments ; core filaments ; carcinoma ; prostate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix (NM) plays roles of fundamental structural and functional significance as the site of replication, transcription, and RNA processing and transport, acting as an anchor or attachment site for a variety of enzymes and other proteins involved in these activities. We have previously documented that protein kinase CK2 translocates from the cytosol to the nucleus, where it associates preferentially with chromatin and NM, in response to certain growth stimuli. Considering that characteristics of the isolated NM can depend on the procedure employed for its isolation, we compared three standard methods for NM preparation to confirm the association of intrinsic CK2 with this structure. Our data suggest that the method used for isolating the NM can quantitatively influence the measurable NM-associated CK2. However, all three methods employed yielded qualitatively similar results with respect to the stimulus-mediated modulation of NM-associated CK2, thus further supporting the notion that NM is an important site for physiologically relevant functions of CK2. In addition, core filaments and cytoskeleton that were isolated by two of the preparative methods had a small but significant level of associated CK2 activity. J. Cell. Biochem. 64:499-504. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 514-523 
    Details
    ISSN: 0730-2312
    Keywords: signal transduction ; stomach ; hormones ; phospholipase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a 〉 two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514-523. © 1997 Wiley-Liss, Inc.
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    Inhibition of human immunodeficiency virus type 1 replication by a cellular transcriptional factor MBP-1 (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 565-572 
    Details
    ISSN: 0730-2312
    Keywords: transcriptional regulation ; HIV-1 ; replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A cellular transcriptional factor initially identified as the c-myc promoter binding protein (MBP-1) was subsequently characterized as a cell regulatory protein with multifunctional activities. In this study, the role of MBP-1 on human immunodeficiency virus type-1 (HIV-1) transcriptional activity was investigated. MBP-1 showed inhibition of HIV-1 long terminal repeat (LTR)-directed chloramphenicol acetyl transferase (CAT) activity in a transient cotransfection assay. Deletion of upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kB) and Sp1 binding sites, did not affect the MBP-1 mediated suppression of HIV-1 LTR. The core promoter of the HIV-1 appeared to be the primary sequence involved in MBP-1 mediated inhibition. In the presence of HIV-1 TAR sequence and Tat protein, MBP-1 did not inhibit the viral promoter activity. In addition, cotransfection experiments with HIV-1 LTR and deletion mutants of MBP-1 suggested that the carboxyl terminal half of MBP-1 suppresses the HIV-1 promoter activity. Exogenous expression of MBP-1 showed suppression of HIV-1 replication in acutely infected cells and in cells cotransfected with a molecular clone of HIV-1. These results suggest that exogenous expression of MBP-1 plays an important role in the regulation of HIV-1 replication in infected cells. J. Cell. Biochem. 64:565-572. © 1997 Wiley-Liss, Inc.
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    Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 408-419 
    Details
    ISSN: 0730-2312
    Keywords: chondrocytes ; calcium ; calmodulin ; binding proteins ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E2 (PGE2) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE2 provoked a 2.7 ± 0.3- and 3.8 ± 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca++ channel blocker, verapamil, and the Ca++/calmodulin inhibitor, W-7. The Ca++ ionophore, ionomycin, mimicked the effects of PGE2. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE2 secretagogue, interleukin-1β (IL-β). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE2 synthesis, down-regulated control, constitute levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca++ and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE2 activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes. J. Cell. Biochem. 65:408-419. © 1997 Wiley-Liss, Inc.
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    Differentiation of human trophoblast cells in vitro is inhibited by dimethylsulfoxide (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 460-468 
    Details
    ISSN: 0730-2312
    Keywords: placenta ; planar-polar compounds ; hCG ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Dimethyl sulfoxide (DMSO) exerts a number of biological effects, the most frequently cited being induction of cell differentiation. The compound also increases invasiveness and metastatic potential. In contrast to the many reports of DMSO-induced cell differentiation, we report here that DMSO inhibits the morphological differentiation of human cytotrophoblast cells to syncytiotrophoblast, as revealed by immunofluorescence staining for desmosomal protein and nuclei. Cytotrophoblast cells treated with DMSO under differentiation-inducing conditions remained mononucleated with intense desmosomal staining. The effect was dose dependent, with a maximal effect seen at 1.5% DMSO. Concentrations of ≤0.5% had no effect and concentrations 〉2% were cytotoxic. In addition to these morphological changes, DMSO inhibited secretion of human chorionic gonadotropin in a dose-dependent manner. At a concentration of 1.5%, DMSO inhibited secretion by 70%. If cytotrophoblast cells were cultured in the presence of DMSO and then switched to DMSO-free medium, they proceeded to differentiate normally. While the precise mechanism of action remains unknown, judicious use of DMSO may be a useful tool for studying and manipulating the differentiation of human trophoblast cells in vitro. The findings also indicate that care should be used in interpreting results obtained using DMSO as a carrier in drug and inhibitor studies. J. Cell Biochem. 65:460-468. © 1997 Wiley-Liss Inc.
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    Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 198-208 
    Details
    ISSN: 0730-2312
    Keywords: tumor necrosis factor-alpha ; G protein ; phosphatidylinositol-specific phospholipase C ; protein kinases ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F4-, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4- -promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate; and a 36% increase in [3H]inositol-1-phosphate within 5 min in prelabeled cells. [3H]inositol-4-phosphate, a metabolite of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F4- -activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F4- by 90-100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F4--induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C. J. Cell. Biochem. 65:198-208. © 1997 Wiley-Liss, Inc.
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    Differential expression of c-jun and junD in end-stage human cardiomyopathy (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 245-253 
    Details
    ISSN: 0730-2312
    Keywords: c-jun ; junD ; cardiomyopathy ; myosin ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The proto-oncogenes c-jun and junD are closely related transcriptional factors with opposing actions on cell growth and division. Expression of c-jun rapidly increases as cells enter the cell cycle. Levels of c-jun are also increased in the early stages of experimental cardiac hypertrophy and failure but expression decreases with time. In contrast, junD accumulates in quiescent cells. Expression in end-stage cardiomyopathy has not been studied. Steady-state levels of c-jun and junD mRNA were determined in failing human myocardium (obtained at the time of cardiac transplantation) and in control myocardium from patients who died of noncardiac causes. Relative expression was normalized for glyceraldehyde-3-phosphate dehydrogenase expression. Levels of junD were almost four-fold depressed in myocardium from myopathic hearts (2.1 ± 0.27, × ± SE; n = 20) vs. the controls (7.7 ± 1.1; n = 3). Levels of c-jun were similar in both myopathic and control hearts. Relative expression of beta-myosin heavy chain was the same in both myopathic and control hearts. Levels of junD were still found to be depressed in the myopathic hearts after normalization for myosin heavy chain gene expression. We conclude that c-jun and junD are differentially regulated in end-stage human cardiomyopathy with expression of junD being decreased while relative levels of c-jun mRNA remain unchanged. Further studies are needed to determine the role of junD down-regulation in the development and/or maintenance of the abnormalities present in end-stage heart disease. J. Cell. Biochem. 65:245-253. © 1997 Wiley-Liss, Inc.
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    Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 9-15 
    Details
    ISSN: 0730-2312
    Keywords: breast cancer ; well/poorly differentiated human breast cancer cells ; estrogen receptor ; nuclear matrix proteins ; diagnostic indicators ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type-specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1-5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9-15, 1997. © 1997 Wiley-Liss, Inc.
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    Studies on the function of Rho A protein in cardiac myofibrillogenesis (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 43-53 
    Details
    ISSN: 0730-2312
    Keywords: rho A ; C3 exoenzyme ; focal adhesion ; costamere ; myofibrillogenesis ; cardiomyocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, β-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and α-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes. J. Cell. Biochem. 66:43-53, 1997. © 1997 Wiley-Liss, Inc.
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    Heat shock protein 27 stimulates recovery of RNA and protein synthesis following a heat shock (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 153-164 
    Details
    ISSN: 0730-2312
    Keywords: thermotolerance ; molecular chaperone ; breast cancer and CHO cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Constitutive expression of human hsp27 resulted in a 100-fold increase in survival to a single lethal heat shock in CHO cells without effecting the development of thermotolerance. A possible mechanism for the thermoprotective function of hsp27 may be increased recovery of protein synthesis and RNA synthesis following a heat shock. A lethal heat shock (44°C, 30 min) results in a 90% reduction in the rate of protein synthesis in non-tolerant cells. Control transfected cells recovered protein synthesis to a pre-heat shock rate 10 h after the heat shock; while cell lines that constitutively express human hsp27 recovered 6 h after the heat shock. Thermotolerant cells had a 50% reduction in protein synthesis, which recovered within 7 h following the heat shock. The same lethal heat shock (44°C, 30 min) reduced RNA synthesis by 60% in the transfected cell lines, with the controls recovering in 7 h; while the hsp27 expressing cell lines recovered within 5 h. Thermotolerant cells had a 40% reduction in RNA synthesis and were able to recover within 4 h. The enhanced ability of hsp27 to facilitate recovery of protein synthesis and RNA synthesis following a heat shock may provide the cell with a survival advantage. J. Cell. Biochem. 66:153-164, 1997. © 1997 Wiley-Liss Inc.
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    Expression of the vitamin D and the retinoid X receptors in Saccharomyces cerevisiae: Alternative in vivo models for ligand-induced transactivation (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 184-196 
    Details
    ISSN: 0730-2312
    Keywords: vitamin D receptor ; retinoid X receptor ; transactivation systems ; vitamin D regulation ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The transcription factors of the nuclear hormone receptor familiy regulate gene expression via a complex network of macromolecular interactions. The ligand dependent activity of the vitamin D receptor is of particular interest because it modulates gene expression by the heterodimeric interaction with retinoid X receptors. We report here that individual functions of the vitamin D receptor including DNA-binding, homo- and heterodimerization and transactivation can be reconstituted in the yeast Saccharomyces cerevisiae. Interestingly, the simultaneous expression of the native vitamin D receptor and the retinoid X receptor β resulted in a ligand independent transactivation of the lacZ reporter gene coupled to a mouse osteopontin vitamin D response element. However, homodimerization of the vitamin D receptor and heterodimerization were strongly enhanced upon ligand binding, when the receptors were expressed as fusion proteins with the Gal4 transcription factor in a yeast two-hybrid system. Furthermore, transactivating activity of a Gal4-fused vitamin D receptor was induced by vitamin D in a one-hybrid system devoid of retinoid X receptors. In addition, both Gal4-based systems behaved similar with regard to their dose-dependent response to vitamin D and related compounds when compared to the transcriptional activity of the vitamin D receptor in transiently transfected MCF-7 cells. Our results point out that specific ligands strongly enhanced receptor dimerization and induced transactivation in yeast and in MCF-7 cells. The constitutive transactivation by vitamin D receptor-retinoid X receptor heterodimers in yeast, depending on DNA binding of the receptors, strongly argues for the existence of cofactors, which are absent in yeast, but play a fundamental role in gene regulation in higher eukaryotic organisms. J. Cell. Biochem. 66:184-196, 1997. © 1997 Wiley-Liss, Inc.
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    Expression of human p140trk receptors in p140trk-deficient, PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 229-244 
    Details
    ISSN: 0730-2312
    Keywords: nerve growth factor ; fibroblast growth factor ; K-252a ; staurosporine ; p140trk ; receptor ; signal transduction ; tyrosine kinase ; transfection ; overexpression ; PC12/endothelial hybrid cells ; DNA synthesis ; proliferation ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal-mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN-trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN-trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates. J. Cell. Biochem. 66:229-244. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Heparan sulfate-binding peptide promotes the deposition of proteoglycans in the extracellular matrix (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 574-590 
    Details
    ISSN: 0730-2312
    Keywords: endothelial cells ; tissue factor pathway inhibitor (TFPI) ; heparan sulfate proteoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A synthetic peptide, which was shown to bind extracellular matrix heparan sulfate chains with a high degree of affinity and specificity [Colburn et al. (1996): Arch Biochem Biophys 325:129-138], has now been found to promote the transfer and the deposition of endothelial cell surface proteoglycans in the extracellular matrix. The peptide also induces preferential binding of extracellular matrix heparan sulfate proteoglycans, which have been added to the supernatant growth medium, and the requirement for its presence is stringent in that only a negligible amount of proteoglycans are bound to the cell layer in the absence of the peptide. In addition, antibodies directed against the peptide detect the accumulation of the peptide in the matrix compartment where the peptide is found associated with the proteoglycans transferred from the cell surface.The sequence of events induced by the peptide appears to be an extension of a naturally occurring process since proteoglycans with properties similar to those of the species ordinarily present in the extracellular matrix have been observed to transfer from the cell surface to the matrix during a pulse-chase experiment. We suggest that formation of the complex peptide-proteoglycan with consequent displacement of the proteoglycan from its anchorage on the cell, initiates the process of transfer of the heparan sulfate-bound peptide from the cell surface to the extracellular matrix. J. Cell. Biochem. 65:574-590. © 1997 Wiley-Liss Inc.
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    ADPribosylation reaction by free ADPribose in Sulfolobus solfataricus, a thermophilic archaeon (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 37-42 
    Details
    ISSN: 0730-2312
    Keywords: archaeon ; ADPribose ; glycation ; ADPribose transferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the archaeon Sulfolobus solfataricus, protein ADPribosylation by free ADPribose was demonstrated by testing both [adenine-14C(U)]ADPR and [adenine- 14C(U)]NAD as substrates. The occurrence of this process was shown by using specific experimental conditions. Increasing the incubation time and lowering the pH of the reaction mixture enhanced the protein glycation by free ADPribose. At pH 7.5 and 10 min incubation, the incorporation of free ADPribose into proteins was highly reduced. Under these conditions, the autoradiographic pattern showed that, among the targets of ADPribose electrophoresed after incubation with 32P-NAD, the proteins modified by free 32P-ADPribose mostly corresponded to high molecular mass components. Among the compounds known to inhibit the eukaryotic poly-ADPribose polymerase, only ZnCl2 highly reduced the ADPribose incorporation from NAD into the ammonium sulphate precipitate. A 20% inhibition was measured in the presence of nicotinamide or 3-aminobenzamide. No inhibition was observed replacing NAD with ADPR as substrate. J. Cell. Biochem. 66: 37-42, 1997. © 1997 Wiley-Liss, Inc.
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    Selective effects of hydrocortisone on intestinal lipoprotein and apolipoprotein synthesis in the human fetus (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 65-76 
    Details
    ISSN: 0730-2312
    Keywords: chylomicron ; very low density lipoprotein ; high density lipoprotein ; apoprotein B-100 ; apoprotein B-48 ; apoprotein A-I ; fat transport ; ontogeny ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Studies employing human fetal intestine have yielded much interesting information on the role of polarized enterocytes in fat absorption and transport. Using the organ culture model, we examined the influence of hydrocortisone on the synthesis and secretion of lipids and lipoproteins. Human jejunal explants were cultured for 5 days at 37°C in serum-free medium containing either [14C]-oleic acid or [14C]-acetate, alone or supplemented with hydrocortisone (25 or 50 ng/ml). The uptake of [14C]-oleic acid was associated with the production of triglycerides, phospholipids, and cholesteryl esters, which were all affected by hydrocortisone. This hormonal agent (50 μg) led to the marked reduction of secreted triglycerides (43%, P 〈 0.01), phospholipids (39%, P 〈 0.01), and cholesteryl esters (36%, P 〈 0.05) without altering the characteristic distribution of tissue and medium lipid classes. Similarly, hydrocortisone significantly (P 〈 0.01) decreased (∼60%) the incorporation of [14C]-acetate into secreted free and esterified cholesterol in the medium. With [14C]-oleic acid as a precursor, hydrocortisone significantly diminished the delivery of chylomicrons and very low density lipoproteins to the medium while consistently enhancing the secretion of high density lipoproteins. In parallel, [35S]-methionine pulse-labeling of jejunal explants revealed the concomitant inhibitory effect of hydrocortisone on apo B-100 synthesis and hydrocortisone's stimulatory effect on apo B-48 and apo A-I. These studies suggest that glucocorticoids play a critical role in lipoprotein processing during intestinal development. J. Cell. Biochem. 66:65-76 1997. © 1997 Wiley-Liss, Inc.
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    Deletion analysis of ors12, a centromeric, early activated, mammalian origin of DNA replication (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 87-97 
    Details
    ISSN: 0730-2312
    Keywords: deletion mutants ; ors12 ; replication activity ; mammalian origin ; autonomous replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized. J. Cell. Biochem. 66:87-97, 1997. © 1997 Wiley-Liss, Inc.
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    Subcellular partitioning of transcription factors during osteoblast differentiation: Developmental association of the AML/CBFα/PEBP2α-related transcription factor-NMP-2 with the nuclear matrix (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 123-132 
    Details
    ISSN: 0730-2312
    Keywords: AML-3 ; transcription factors ; partitioning ; osteoblast differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, we systematically analyzed the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) during osteoblast differentiation using biochemical fractionation and gel shift analyses. We show that nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. Moreover, we show that there are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. We conclude that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. Our results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype. J. Cell. Biochem. 66:123-132, 1997. © 1997 Wiley-Liss, Inc.
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    Polyamine-dependent alterations in the structure of microfilaments, golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 165-174 
    Details
    ISSN: 0730-2312
    Keywords: cell shape ; cytoskeleton ; stress fibers ; autophagy ; vacuolar degradation ; hyaluronan ; chondroitin sulfate ; keratan sulfate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [3H]glucosamine and [35S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165-174, 1997. © 1997 Wiley-Liss, Inc.
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    Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 219-228 
    Details
    ISSN: 0730-2312
    Keywords: DNA strand breaks ; superoxide ; granulocytes ; human ; okadaic acid ; fluoride ; staurosporine ; phorbol myristate acetate ; hydrogen peroxide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Phorbol ester treatment of granulocytes triggers release of superoxide (O2-) and a concomitant burst of DNA strand breaks. The relationship between the amount of O2- and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O2- generated over a 40-min period by 1 × 106 granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O2- per 1 × 106 cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O2- released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1-10 mM) and staurosporine (2-10 nM) both inhibited O2- production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O2-. Zinc chloride (50-200 μM) inhibited both O2- and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O2- production more effectively than it inhibited DNA breaks. O2- dismutes to H2O2, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H2O2 had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O2- or H2O2 in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219-228, 1997. © 1997 Wiley-Liss Inc.
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    Human papillomavirus (HPV) 16 E6 sensitizes cells to atractyloside-induced apoptosis: Role of p53, ICE-like proteases and the mitochondrial permeability transition (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 245-255 
    Details
    ISSN: 0730-2312
    Keywords: p53 ; HPV ; apoptosis ; mitochondrial permeability transition ; ICE-like proteases ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Infection of cervical epithelial cells with certain high risk HPV genotypes is thought to play an etiologic role in the development of cervical cancer. In particular, HPV type 16 and 18 early protein 6 (E6) is thought to contribute to epithelial transformation by binding to the tumor suppressor protein p53, targeting it for rapid proteolysis, resulting in loss of its cell cycle arrest and apoptosis-inducing activities. Recent data indicate that factors responsible for triggering apoptosis reside in the cytoplasm of cells, and not in the nucleus. In particular, the findings that mitochondria are required in certain cell-free models for induction of apoptosis and that bcl-2 is localized to mitochondria have focused attention on the role of the mitochondrial membrane permeability transition (MPT) in apoptosis. Here we present data to indicate that HPV 16 E6 expression sensitizes cells to MPT-induced apoptosis. We also report that HPV 16 E6 sensitization of cells to MPT-induced apoptosis occurs only in the presence of wildtype (wt) p53 expression. The extent of apoptosis induced by atractyloside (an inducer of the MPT) in normal, temperature-sensitive (ts) p53, and HPV-16 E6 transfected J2-3T3 cells, and the HPV expressing cervical carcinoma cell lines SiHa, Hela and CaSki was determined. C33A cells, which express mutant p53 but not HPV, were also exposed to atractyloside in the presence or absence of HPV 16 E6 expression. Dose-dependent apoptosis induced by atractyloside in normal J2-3T3 cells and cervical carcinoma cells was measured by loss of cell viability, nuclear fragmentation and DNA laddering. The sensitivity of cells to atractyloside-induced apoptosis was found to be: HPV 16 E6-J2-3T3 〉 CaSki 〉 normal-J2-3T3 cells ≈ ts p53-J2-3T3 ≈ vector-J2-3T3 cells 〉 Hela 〉 SiHa 〉 C33A ≈ C33A 16 E6. Cyclosporin A (CsA), an inhibitor of the MPT, and ICE-I, a protease inhibitor, provided protection against atractyloside-induced apoptosis. These findings indicate that: 1) high risk HPV 16 E6 protein is capable of sensitizing cells to apoptosis; 2) HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis occurs in a p53-dependent fashion; 3) the target of HPV 16 E6 sensitization of cells to atractyloside-induced apoptosis is the mitochondria; and 4) HPV 16 E6 sensitization of cells to atroctycoside-induced apoptosis involves an ICE-like protease-sensitive mechanism, regulating the onset of the MPT. These findings constitute the first evidence that mitochondria play a role in HPV 16 E6 modulation of apoptosis. J. Cell. Biochem. 66:245-255. © 1997 Wiley-Liss, Inc.
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    Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 309-321 
    Details
    ISSN: 0730-2312
    Keywords: oncogenes ; tumor suppressors ; human papillomavirus type 16 ; smoking cofactor ; immortalization ; tumorigenesis ; mRNA ; proteins ; oncogenesis ; senescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis. J. Cell. Biochem. 66: 309-321, 1997. © 1997 Wiley-Liss, Inc.
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    Characterization of a metalloproteinase: A late stage specific gelatinase activity in the sea urchin embryo (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 337-345 
    Details
    ISSN: 0730-2312
    Keywords: sea urchin ; embryo ; gelatinase ; metalloproteinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have partially purified and characterized an 87 kDa gelatinase activity expressed in later stage sea urchin embryos. Cleavage activity was specific for gelatin and no cleavage of sea urchin peristome type I collagen, bovine serum albumin or casein was detected. Magnesium and Zn2+ inhibited the gelatinase and Ca2+ protected against inhibition. Ethylenediamine tetracetic acid, ethylenebisoxyethylenenitriol tetraacetic acid and 1,10-phenanthroline were inhibitory, suggesting that the gelatinase is a Ca2+- and Zn2+-dependent metalloproteinase. No inhibition was detected with serine or cysteine protease inhibitors and the vertebrate matrix metalloproteinase (MMP) inhibitor, Batimastat, was also ineffective. The vertebrate MMP activator p-aminophenylmercuric acetate was without effect. These results allow us to identify both similarities and differences between echinoderm and vertebrate gelatinases. J. Cell. Biochem. 66: 337-345, 1997. © 1997 Wiley-Liss, Inc.
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    Kips off to Myc: Implications for TGFβ signaling (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 427-432 
    Details
    ISSN: 0730-2312
    Keywords: TGFβ ; transforming growth factor β ; Cdk ; cyclin-dependent kinase ; Kip ; cdk-inhibitor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Loss of sensitivity to the negative growth regulator transforming growth factor β (TGFβ) is a feature of many different tumor types and is likely involved in tumor progression. In some cases this loss of sensitivity to TGFβ has been shown to be manifest in the absence of membrane-associated TGFβ receptor complexes, thus preventing initiation of antiproliferative signals from the cell surface. In others, loss of sensitivity to TGFβ-induced inhibitory signals has been attributed to loss of function of intracellular effectors of TGFβ-induced inhibitory signals due to mutation or allelic loss of effector genes and their products. The intracellular effectors of TGFβ inhibitory signals have been shown to be involved in the normal regulation of progression through the cell cycle, specifically during G1 phase. In this manner, elucidation of the mechanisms by which TGFβ inhibits cell growth not only helps us identify steps involved in tumor progression, but also allows us to better understand how cells regulate progression through the cell cycle. J. Cell. Biochem. 66:427-432, 1997. © 1997 Wiley-Liss, Inc.
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    Heat stress induces tyrosine phosphorylation/activation of kinase Fa/GSK-3α (a human carcinoma dedifferentiation modulator) in A431 cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 16-26 
    Details
    ISSN: 0730-2312
    Keywords: heat stress ; kinase FA/GSK-3&agr ; tyrosine phosphorylation/activation ; cascade activation ; protein expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Exposure of A431 cells to a rapid temperature increase from 37° to 46°C could induce an increased expression (∼200% of control) and tyrosine phosphorylation/activation (∼300% of control) of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) in a time-dependent manner, as demonstrated by an anti-kinase FA/GSK-3α immunoprecipitate kinase assay and by immunoblotting analysis with anti-kinase FA/GSK-3α and anti-phosphotyrosine antibodies. The heat induction on the increased expression of kinase FA/GSK-3α could be blocked by actinomycin D but not by genistein. In contrast, the heat induction on tyrosine phosphorylation/activation of kinase FA/GSK-3α could be blocked by genistein or protein tyrosine phosphatase, indicating that heat stress induces a dual control mechanism, namely, protein expression and subsequent tyrosine phosphorylation to cause cellular activation of kinase FA/GSK-3α. Taken together, the results provide initial evidence that kinase FA/GSK-3α represents a newly described heat stress-inducible protein subjected to tyrosine phosphorylation/activation, representing a new mode of signal transduction for the regulation of this human carcinoma dedifferentiation modulator and a new mode of heat induction on cascade activation of a protein kinase. J. Cell. Biochem. 66:16-26, 1997. © 1997 Wiley-Liss, Inc.
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    Calpain activation in shear-induced platelet aggregation (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 54-64 
    Details
    ISSN: 0730-2312
    Keywords: calpain activation ; platelet ; proteolysis of talin ; shear stress ; shear-induced platelet aggregation (SIPA) ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm2 was applied to platelets, activation of μ-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm2, μ-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm2, applied for 5 min, caused μ-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both μ-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization. J. Cell. Biochem. 66:54-64, 1997. © 1997 Wiley-Liss, Inc.
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    Cell cycle-dependent modifications in activities of pRb-related tumor suppressors and proliferation-specific CDP/cut homeodomain factors in murine hematopoietic progenitor cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 512-523 
    Details
    ISSN: 0730-2312
    Keywords: cell cycle control ; H4 gene promoter ; G1/S phase transition point ; CDP/cut ; interferon regulatory factor 2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase, and the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the homeodomain CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, we have investigated these determinants of transcriptional regulation at the G1/S phase transition in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. We find striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. We also observe a dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal. J. Cell. Biochem. 66:512-523, 1997. © 1997 Wiley-Liss, Inc.
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    Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 248-256 
    Details
    ISSN: 0730-2312
    Keywords: human prostatic cancer cell (PC-3) ; osteoblastic cell differentiation ; bone nodule formation ; alkaline phosphatase activity ; osteocalcin ; osteopontin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5-30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248-256, 1997. © 1997 Wiley-Liss, Inc.
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    Retinoblastoma-related protein pRb2/p130 and its binding to the B-myb promoter increase during human neuroblastoma differentiation (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 297-303 
    Details
    ISSN: 0730-2312
    Keywords: retinoblastoma family ; pRb ; p107 ; pRb2/p130 ; neuroblastoma ; differentiation ; B-myb ; c-myb ; E2F ; promoter ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins. J. Cell. Biochem. 67:297-303, 1997. © 1997 Wiley-Liss, Inc.
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    Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 338-352 
    Details
    ISSN: 0730-2312
    Keywords: basement membrane ; vitamin C ; degradation ; proline hydroxylation ; teratocarcinoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator α,α′-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2-4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by α,α′-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (- ascorbate) hydroxylated α1(IV) or α2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15°C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion. J Cell. Biochem. 67:338-352, 1997. Published 1997 Wiley-Liss, Inc.
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    Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2 (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 386-398 
    Details
    ISSN: 0730-2312
    Keywords: human osteoblasts ; human bone marrow stromal cells ; alkaline phosphatase ; osteopontin ; bone sialoprotein ; osteonectin ; decorin ; biglycan ; type I collagen ; osteocalcin ; mineralization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of β-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1-7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386-398, 1997. © 1997 Wiley-Liss, Inc.
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    Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 439-450 
    Details
    ISSN: 0730-2312
    Keywords: artificial chromosome ; episome ; YAC ; nuclear matrix attachment region ; MAR ; replication origin ; DNA replication ; fluorescent in situ hybridization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes. J. Cell. Biochem. 67:439-450, 1997. © 1997 Wiley-Liss, Inc.
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    α2-Macroglobulin synthesis by the human monocytic cell line THP-1 is differentiation state-dependent (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 492-497 
    Details
    ISSN: 0730-2312
    Keywords: interferon-γ ; PMA ; proteinase inhibitor ; cytokine ; low density lipoprotein receptor-related protein ; receptor-associated protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human α2-macroglobulin (α2M) is a broad spectrum proteinase inhibitor and cytokine carrier synthesized by a number of cell types including monocytes and macrophages. In this study, we report on the expression of α2M by THP-1 cells. This monocytic cell line can be differentiated into a macrophage-like phenotype by treatment with interferon-γ (IFN-γ) or phorbol 12-myristate 13-acetate (PMA). α2M was synthesized by THP-1 cells at a rate of 75 ng/106 cells/24 h, as determined by Western blot analysis. After treating the cells with 500 U/ml of IFN-γ or with 100 ng/ml PMA, the synthesis rate increased to 219 ng/106 cells/24 h and to 179 ng/106 cells/24 h, respectively. The same agents also increased α2M expression, as determined by Northern blot analysis. When the α2M receptor antagonist, receptor associated protein (RAP), was included in the THP-1 medium, the amount of α2M recovered in the conditioned medium increased. This result suggests that THP-1-secreted proteinases react with secreted α2M and that the resulting complexes are catabolized by the α2M receptor, which is also called low density lipoprotein receptor-related protein (LRP). We conclude that α2M synthesis by THP-1 cells depends on the state of cellular differentiation. Reaction of α2M with secreted proteinases may have minimized previous estimates of the rate of synthesis of α2M by certain cells in culture. J. Cell. Biochem. 67:492-497, 1997. © 1997 Wiley-Liss, Inc.
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    Actin isoform utilization during differentiation and remodeling of BC3H1 myogenic cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 514-527 
    Details
    ISSN: 0730-2312
    Keywords: smooth muscle ; actin ; myogenesis ; cytoskeleton ; microfilaments ; protein crosslinking ; muscle cells ; cell fractionation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mouse BC3H1 myogenic cells and a bi-functional chemical cross linking reagent were utilized to investigate the polymerization of newly-synthesized vascular smooth muscle (α-actin) and non-muscle (β- and γ-actin) actin monomers into native F-actin filament structures during myogenesis. Two actin dimer species were identified by SDS-PAGE analysis of phenylenebismaleimide-cross linked fractions of BC3H1 myoblasts and myocytes. P-dimer was derived from the F-actin-enriched, detergent-insoluble cytoskeleton. Pulse-chase analysis revealed that D-dimer initially was associated with the cytoskeleton but then accumulated in the soluble fraction of lysed muscle cells that contained a non-filamentous or aggregated actin pool. Immunoblot analysis indicated that non-muscle and smooth muscle actins were capable of forming both types of dimer. However, induction of smooth muscle α-actin in developing myoblasts coincided with an increase in D-dimer level which may facilitate actin stress fiber assembly. Smooth muscle α-actin was rapidly utilized in differentiating myoblasts to assemble extraction-resistant F-actin filaments in the cytoskeleton whereas non-muscle β- and γ-actin filaments were more readily dissociated from the cytoskeleton by an extraction buffer containing ATP and EGTA. The data indicate that cytoarchitectural remodeling in developing BC3H1 myogenic cells is accompanied by selective actin isoform utilization that effectively segregates multiple isoactins into different sub-cellular domains and/or supramolecular entities. J. Cell. Biochem. 67:514-527, 1997. © 1997 Wiley-Liss, Inc.
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    Overview (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. v 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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    Matrix attachment regions and transcription units in a polygenic mammalian locus overlapping two isochores (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 541-551 
    Details
    ISSN: 0730-2312
    Keywords: chromatin loops ; chromosome organization ; compositional mapping ; gene cluster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Eukaryotic chromosomes are ponctuated by specialized DNA sequences (MARs) characterized by their ability to bind the network of nonhistone proteins that form the nuclear matrix or scaffold. We previously described an amplifiable cluster of genes with different tissue-specific expression patterns, located on Chinese hamster chromosome 1q. This model is especially appropriate to study the relationships between MARs and transcription units. We show here that four attachment regions, with sequences exhibiting motifs specific to MARs, are present within the 100 kb of screened DNA. Three of them are relatively short sequences localized in intergenic regions. The last one extends over one of the transcription units and contains a region previously identified as a recombination hot spot. Moreover, the analysis of a DNA sequence extending over some 50 Kb of this region and spanning at least four genes, disclosed a strikingly sharp change in G + C content. This strongly suggests that the studied region contains the boundary of two isochores. We propose that the frequency and the size of MARs are correlated to their localization in G + C rich or poor domains. J. Cell. Biochem. 67:541-551, 1997. © 1997 Wiley-Liss, Inc.
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    Chemopreventive effect of perillyl alcohol on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced tumorigenesis in (C3H/HeJ X A/J)F1 mouse lung (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 20-25 
    Details
    ISSN: 0730-2312
    Keywords: CAAX motif ; farnesyltransferase inhibitor ; K-ras ; lung cancer ; monoterpene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: This study was designed to test the chemopreventive potential of perillyl alcohol, an inhibitor of farnesyltransferase, in a mouse lung tumor bioassay. Perillyl alcohol is a naturally occurring monoterpene found in lavender, cherries, and mint. We have shown previously that the majority of lung tumors in this bioassay have an activating mutation in the K-ras gene, which occurs early in the development of mouse lung carcinogenesis. The Ras protein undergoes a series of post-translational modifications, the first of which is farnesylation at the cysteine of the C-terminal CAAX motif. These modifications lead to the anchoring of Ras p21 to the plasma membrane in its biologically active state. Activated Ras p21 couples growth regulatory signals from receptor tyrosine kinases to cytoplasmic second messengers. In a preliminary study, we determined the maximum tolerated dose of perillyl alcohol to be 75 mg/kg body weight. For the bioassay, 5-week-old male (C3H/HeJ X A/J) F1 hybrid mice were randomized into trial groups, and treated with perillyl alcohol three times per week i.p., starting 1 week prior to initiation with the carcinogen NNK, and continuing for 22 weeks after initiation. Our results show a 22% reduction in tumor incidence, and a 58% reduction in tumor multiplicity. Our study demonstrates that perillyl alcohol is an effective chemopreventive compound in the mouse lung tumor bioassay. J. Cell. Biochem. Suppl. 27:20-25. © 1998 Wiley-Liss, Inc.
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    Influence of tea catechins on the digestive tract (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 52-58 
    Details
    ISSN: 0730-2312
    Keywords: tea ; green tea ; tea polyphenols ; Polyphenon® ; tea catechins ; EGCg ; fecal flora ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Tea catechins undergo various metabolic changes after they are taken orally, though a large percentage are excreted intact with the feces. Epidemiological studies suggest a protective effect of tea against various human cancers, including colon and rectum. The bactericidal property of tea catechins plays several roles in the digestive tract. In the small intestine, catechins inhibit α-amylase activity, and a certain amount is absorbed into the portal vein. Although catechins are bactericidal, they do not affect lactic acid bacteria. Including tea catechins in the diet for several weeks decreases putrefactive products and increases organic acids by lowering pH. These changes were achieved in tube-fed patients by administering 100 mg of tea catechins (equivalent to a cup of green tea) three times daily with meals for 3 weeks. When catechin administration ceased, the effects reversed after 1 week. Catechins should be considered further in colon carcinogenesis studies. J. Cell. Biochem. Suppl. 27:52-58. © 1998 Wiley-Liss, Inc.
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    Tea antioxidants in cancer chemoprevention (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 59-67 
    Details
    ISSN: 0730-2312
    Keywords: antioxidants ; black tea ; chemoprevention ; epigallocatechin-3-gallate ; green tea ; tea polyphenols ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In recent years, the concept of cancer chemoprevention has matured greatly. Significant reversal or suppression of premalignancy in several sites by chemopreventive agents appears achievable. This article summarizes experimental data on chemopreventive effects of tea polyphenols in different tumor bioassay systems. Tea (Camellia sinensis) is cultivated in about 30 countries, and is the most widely consumed beverage in the world. Three main commercial tea varieties - green, black, and oolong - are usually consumed, but most experimental studies demonstrating the antimutagenic and anticarcinogenic effects of tea have been conducted with water extract of green tea, or a polyphenolic fraction isolated from green tea (GTP). The majority of these studies have been conducted in a mouse skin tumor model system where tea is fed either as water extract through drinking water, or as purified GTP. GTP has been shown to exhibit antimutagenic activity in vitro, and inhibit carcinogen- as well as UV-induced skin carcinogenesis in vivo. Tea consumption has also been shown to afford protection against chemical carcinogen-induced stomach, lung, esophagus, duodenum, pancreas, liver, breast, and colon carcinogenesis in specific bioassay models. Several epicatechin derivatives (polyphenols) present in green tea have been shown to possess anticarcinogenic activity; the most active is (-)-epigallocatechin-3-gallate, which is also the major constituent of GTP. The mechanisms of tea's broad cancer chemopreventive effects are not completely understood. Several theories have been put forward, including inhibition of UV- and tumor promoter-induced ornithine decarboxylase, cyclo-oxygenase, and lipoxygenase activities, antioxidant and free radical scavenging activity; enhancement of antioxidant (glutathione peroxidase, catalase, and quinone reductase) and phase II (glutathione-S-transferase) enzyme activities; inhibition of lipid peroxidation, and anti-inflammatory activity. These properties of tea polyphenols make them effective chemopreventive agents against the initiation, promotion, and progression stages of multistage carcinogenesis. J. Cell. Biochem. Suppl. 27:59-67. Published 1998 Wiley-Liss, Inc.
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    Chemopreventive potential of thiol conjugates of isothiocyanates for lung cancer and a urinary biomarker of dietary isothiocyanates (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 76-85 
    Details
    ISSN: 0730-2312
    Keywords: thiol conjugates ; isothiocyanates ; lung cancer prevention ; urinary biomarker ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Natural and synthetic isothiocyanates (ITCs) are versatile chemopreventive agents in many animal systems. We have shown that phenethyl ITC (PEITC) and 6-phenylhexyl ITC (PHITC) are potent inhibitors against lung tumorigenesis induced by tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in both mouse and rat. The mechanism by which these ITCs inhibited lung tumorigenesis is attributed to their ability to decrease cytochrome P450 (P450) enzyme activities involved in the activation of NNK. Recently, we have found that thiol conjugates of ITCs inhibit P450 enzymes and are effective inhibitors of lung tumorigenesis. This is significant because conjugation with cellular thiols is the major route of ITC metabolism via the mercapturic acid pathway in rodents and humans. The thiol conjugates are less pungent and potentially less toxic, and they are more soluble and chemically less reactive than ITCs. These properties raise the prospect of substituting thiol conjugates for ITCs as chemopreventive agents. Furthermore, although ample rodent studies have established that ITCs inhibit tumorigenesis, the protective role of dietary ITCs against human cancers has not yet been established. As a prerequisite for such human studies, we have developed an HPLC-based assay, based on the condensation reaction of ITCs or conjugates with 1,2-benzenedithiol, for measuring a cyclocondensation product in human urine as an uptake biomarker of total ITCs. This assay was validated using urine samples from subjects who had ingested a known amount of watercress or mustard in a controlled diet. The assay is convenient and rapid, showing promise for analyzing urine samples obtained from population-based studies. Results from two such studies are presented to illustrate the potential application of this biomarker in epidemiologic studies. J. Cell. Biochem. Suppl. 27:76-85. © 1998 Wiley-Liss, Inc.
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    Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 368-387 
    Details
    ISSN: 0730-2312
    Keywords: immortalized ; clonal ; alkaline phosphatase ; osteocalcin ; mineralization ; vitamin D3 ; dexamethasone ; parathyroid hormone ; interleukin-6 ; bone ; osteoporosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ERα mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17β-estradiol (17β-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34°C) but stopped dividing at the nonpermissive temperature (&ge 39°C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1&agr 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-β1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17β-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17β-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (∼ 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17β-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17β-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17β-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone. J. Cell. Biochem. 65:368-387. © 1997 Wiley-Liss, Inc.
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    Opposing effects of tyrosine kinase inhibitors on mineralization of normal and tumor bone cells (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 420-429 
    Details
    ISSN: 0730-2312
    Keywords: osteosarcoma ; osteoprogenitors ; tyrphostins ; marrow-stroma ; quinazoline ; benzylidine-malononitrile ; cell proliferation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Induction of matrix maturation and mineralization in calcified tissues is important for patients with primary bone tumors and other bone deficiencies, e.g., osteoporosis. For the former it signifies a better prognosis in osteosarcoma, and for the latter it might improve bone remodeling. In the present study we exposed osteosarcoma cells (Saos2), normal bone cells, and marrow stroma to two different tyrosine kinase (TK) inhibitors: AG-555 and AG-1478. These tyrphostins differ in their effect on signal transduction downstream to the TK receptor (RTK): AG-1478 inhibits src family TKs whereas AG-555 inhibits nuclear TKs. We found that both tyrphostins at 50 μM increased specific alkaline phosphatase (ALP) activity in Saos2 cells. AG-555 abrogated mineralization whereas AG-1478 increased it. Similarly, in human bone-derived cell cultures the same dose of tyrphostins had an opposing effect on mineralization but, in contrast to AG-555, AG-1478 positively selected cells with ALP activity. These tyrphostins also differed in their effect on rat marrow stromal cells. AG-555 decreased cell counts unselectively, whereas the decreased cell counts by AG-1478 resulted in selection of osteoprogenitor cells as indicated by a concordant increase in specific ALP activity. The effect of a lower dose of AG-1478, 5 μM, on the increase in mineralization exceeded its own efficiency in selecting cells with specific ALP activity. Our results indicate that AG-1478 selects and preserves the osteoblastic phenotype, at doses moderately higher than those required to induce mineralization, and substantially higher than the doses required for RTK inhibition. Identification of downstream molecular targets for AG-1478, in marrow stromal cells, might prove useful in designing more selective drugs, capable of separating proliferative from differentiation-inducing activities. J. Cell. Biochem. 65:420-429. © 1997 Wiley-Liss, Inc.
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    Dexamethasone induces rapid actin assembly in human endometrial cells without affecting its synthesis (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 492-500 
    Details
    ISSN: 0730-2312
    Keywords: dexamethasone ; nongenomic effect ; actin assembly ; signal transduction ; confocal microscopy ; total actin ; actin transcript ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Dexamethasone exerts a stimulatory effect of rapid-onset on the polymerization of actin. This has been documented in human endometrial adenocarcinoma Ishikawa cells, resulting in an acute, dose-dependent decrease in the G/total-actin ratio. In the present study we completely characterized this fast and apparently nongenomic effect of dexamethasone on actin assembly. We followed the morphological alterations of actin cytoskeleton and measured the time-dependent dynamics of actin polymerization both by ruling out any changes of total actin in the cells and by measuring its transcript. Rapid changes in actin polymerization were accurately measured using a highly sensitive and quantitative rhodamine-phalloidin fluorimetric assay. Ishikawa cells, exposed to 0.1 μM dexamethasone for various time periods up to 24 h, showed a highly significant, rapid, and transient increase in the polymerization of actin starting within 15 min of dexamethasone exposure and lasting 2 h. Treated cells showed a significant (1.79-fold) enhancement of the fluorescent signal compared to untreated cells at 15 min. This value decreased continuously in a time-dependent manner, reaching control levels after 120 min and remained so for the next 24 h. Confocal laser scanning microscopy studies confirmed these findings. Intensive coloration of microfilaments over several scanning sections suggested an enhanced degree of actin polymerization in cells preincubated for 15 min with 0.1 μM dexamethasone. Moreover, actin filaments were more resistant to cytochalasin B. Additionally, quantitative immunoblot analysis showed that the content of total cellular actin remained the same during this period, suggesting that the biosynthesis of actin was unaffected. Northern blot analysis showed that the concentration of the actin transcript was also unaffected. Our data suggest that glucocorticoids induce a fast and self-limited polymerization of actin in human endometrial cells without affecting its synthesis. These findings strengthen the hypothesis that glucocorticoids exert rapid, nongenomic cellular effects and that the actin-based cytoskeleton is an integral part of this pathway, playing an essential role in receiving and mediating steroid signals for the modulation of cellular responses. J. Cell. Biochem. 65:492-500. © 1997 Wiley-Liss Inc.
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    Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3 (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 542-549 
    Details
    ISSN: 0730-2312
    Keywords: procollagen synthesis ; human osteosarcoma cells ; 1,25-dihydroxyvitamin D3 ; type I collagen ; proline hydroxylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)2 D3-treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)2 D3-treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen α1 chain by 1,25-(OH)2 D3 treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)2 D3 treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)2 D3 treatment was also found to induce the α subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B-resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)2 D3-treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis. J. Cell. Biochem. 65:542-549. © 1997 Wiley-Liss Inc.
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    Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 1-8 
    Details
    ISSN: 0730-2312
    Keywords: AML/CBF/PEBP2 ; regulatory element ; AML-3 ; osteoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2, and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently, AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2. AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation. J. Cell. Biochem. 66:1-8, 1997. © 1997 Wiley-Liss, Inc.
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    Uncoupling of p21 induction and MyoD activation results in the failure of irreversible cell cycle arrest in doxorubicin-treated myocytes (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 27-36 
    Details
    ISSN: 0730-2312
    Keywords: cell cycle ; p21 ; MyoD ; E2F ; doxorubicin/adriamicin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor Id2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells. J. Cell. Biochem. 66:27-36, 1997. © 1997 Wiley-Liss, Inc.
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    Growth factor regulation of insulin-like growth factor binding protein-6 expression in osteoblasts (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 77-86 
    Details
    ISSN: 0730-2312
    Keywords: skeletal cells ; transforming growth factor &Bgr ; transcripts ; bone formation ; local factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previously we have shown that transforming growth factor β (TGF β) 1, basic fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) BB inhibit the synthesis of insulin-like growth factor (IGF) II, but their effects on IGF binding protein (IGFBP)-6 in osteoblast cultures are not known. IGFBP-6 binds IGF II with high affinity and prevents IGF II-mediated effects, so that a possible mode of regulating the IGF II available to bone cells would be by changing the levels of IGFBP-6. To enhance our understanding of the actions of growth factors on the IGF II axis in bone, we tested the effects of TGF β1, basic FGF, PDGF BB, IGF I, and IGF II on the expression of IGFBP-6 in cultures of osteoblast-enriched cells from 22 day fetal rat calvariae (Ob cells). Treatment of Ob cells with TGF β1 caused a time- and dose-dependent decrease in IGFBP-6 mRNA levels, as determined by Northern blot analysis. The effect was maximal after 48 h and observed with TGF β1 concentrations of 0.04 nM and higher. TGF β1 also decreased IGFBP-6 polypeptide levels in the medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 μM decreased IGFBP-6 transcripts and prevented the effect of TGF β1. The decay of IGFBP-6 mRNA in transcriptionally arrested Ob cells was not modified by TGF β1. In addition, TGF β1 decreased the rates of IGFBP-6 transcription as determined by a nuclear run-on assay. In contrast, basic FGF, PDGF BB, IGF I, and IGF II did not change IGFBP-6 mRNA levels in Ob cells. In conclusion, TGF β1 inhibits IGFBP-6 expression in Ob cells by transcriptional mechanisms. Since IGFBP-6 binds IGF II and prevents its effects on bone cells, decreased synthesis of IGFBP-6 induced by TGF β1 could be a local feedback mechanism to increase the amount of IGF II available in the bone microenvironment. J. Cell. Biochem. 66:77-86, 1997. © 1997 Wiley-Liss, Inc.
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  • 92
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    Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 133-140 
    Details
    ISSN: 0730-2312
    Keywords: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ; RNA binding protein ; DNA replication ; DNA repair ; apoptosis ; triplet repeat neurodegenerative disorders ; nitric oxide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested. J. Cell. Biochem. 66:133-140, 1997. © 1997 Wiley-Liss, Inc.
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  • 93
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    Phosphorylation of the oncogenic transcription factor interferon regulatory factor 2 (IRF2) in vitro and in vivo (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 175-183 
    Details
    ISSN: 0730-2312
    Keywords: phosphorylation ; interferon regulatory factor 2 ; transcription factor ; oncogene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: IRF2 is a transcription factor, possessing oncogenic potential, responsible for both the repression of growth-inhibiting genes (interferon) and the activation of cell cycle-regulated genes (histone H4). Surprisingly little is known about the post-translational modification of this factor. In this study, we analyze the phosphorylation of IRF2 both in vivo and in vitro. Immunoprecipitation of HA-tagged IRF2 expressed in 32P-phosphate labelled COS-7 cells demonstrates that IRF2 is phosphorylated in vivo. Amino acid sequence analysis reveals that several potential phosphorylation sites exist for a variety of serine/threonine protein kinases, including those of the mitogen activated protein (MAP) kinase family. Using a battery of these protein kinases we show that recombinant IRF2 is a substrate for protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CK2) in vitro. However, other serine/threonine protein kinases, including the MAP kinases JNK1, p38, and ERK2, do not phosphorylate IRF2. Two-dimensional phosphopeptide mapping of the sites phosphorylated by PKA, PKC, and CKII in vitro demonstrates that these enzymes are capable of phosphorylating IRF2 at multiple distinct sites. Phosphoaminoacid analysis of HA-tagged IRF2 immunoprecipitated from an asynchronous population of proliferating, metabolically phosphate-labelled cells indicates that this protein is phosphorylated exclusively upon serine residues in vivo. These results suggest that the oncogenic protein IRF2 may be regulated via multiple pathways during cellular growth. J. Cell. Biochem. 66:175-183, 1997. © 1997 Wiley-Liss, Inc.
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  • 94
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    Localization of silencer and enhancer elements in the human type X collagen gene (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 210-218 
    Details
    ISSN: 0730-2312
    Keywords: collagen type X ; gene regulation ; calcium phosphate ; reporter gene ; transfection ; hypertrophic chondrocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5′-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements. J. Cell. Biochem. 66:210-218, 1997. © 1997 Wiley-Liss, Inc.
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  • 95
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    Use of lanthanum to accurately quantify insulin-like growth factor binding to proteins on cell surfaces (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 256-267 
    Details
    ISSN: 0730-2312
    Keywords: zinc ; IGFBP ; IGF ; des-(1-3)-IGF-I ; receptor ; fibroblasts ; glioblastoma ; kidney epithelial cells ; affinity ; T98G ; GM10 ; MDBK ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Insulin-like growth factor binding proteins (IGFBPs) are found both associated with cells and in extracellular fluids. Cell-associated IGFBPs increase [125I]-IGF binding to cell monolayers, whereas extracellular (soluble, released) IGFBPs decrease binding. In the current study, we show that either IGFBP-3 or IGFBP-5 are the major forms of IGFBP released from monolayers of human GM10 fibroblasts, T98G glioblastoma cells and forskolin-treated bovine MDBK cells. IGFBPs represent the most abundant [125I]-IGF-I binding site on GM10 and T98G cell monolayers, but 4-17% of the total cell-associated IGFBPs are released from the cell monolayer at 8°C during their quantification. Most of the IGFBPs (〉 70%) are released from MDBK cells. Quantitative estimates of [125I]-IGF binding to the cell monolayers are altered because of the ability of the released IGFBPs to reduce the amount of radiolabeled ligand that is available to bind to the cell surface. Lanthanum (La3+) depresses IGFBP release from all three cell types (〉 80% for GM10 and T98G cells and 〉 65% for MDBK cells). The effect was cation specific, noted with La3+ or Zn2+ but not with either Mn2+, Sr2+ or Se3+. The effect was also IGFBP specific; La3+ markedly depressed the release of IGFBP-3 and IGFBP-5, but had less of an effect on IGFBP-2 and IGFBP-4. Concomitant with a decrease in IGFBP-3 and IGFBP-5 release, La3+ caused an increase in [125I]-IGF-I binding to cell-associated IGFBPs and type I IGF receptors. The released soluble IGFBPs have a three- to 20-fold greater affinity (Ka) for [125I]-IGF-I compared to cell-associated IGFBPs. La3+ did not alter the affinity constants of cell-associated IGFBPs. In summary, we have identified a means to prevent loss of IGFBPs from cell monolayers during binding assays. This procedure will be useful in accurately quantifying the levels of IGFBPs on cell monolayers and in determining the role of cell-associated IGFBPs in controlling IGF activity. Retention of cell-associated low affinity IGFBPs may be important in controlling the size of the pericellular IGF pool and in regulating IGF-I access to the type I IGF receptor. J. Cell. Biochem. 66:256-267. © 1997 Wiley-Liss, Inc.
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  • 96
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    Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 394-403 
    Details
    ISSN: 0730-2312
    Keywords: hypertrophic ; growth plate ; type X ; alkaline phosphatase ; sternum ; chicken ; avian ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In serum-containing medium, ascorbic acid induces maturation of prehypertrophic chick embryo sternal chondrocytes. Recently, cultured chondrocytes have also been reported to undergo maturation in the presence of bone morphogenetic proteins or in serum-free medium supplemented with thyroxine. In the present study, we have examined the combined effect of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocytes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels within 24-72 h, and simultaneous exposure to both ascorbate and BMP yielded enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and BMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent serum-free cultures treated for 48 h with BMP + ascorbate were equivalent to those reached in serum-containing medium only after confluence. While ascorbate addition was required for maximal alkaline phosphatase activity, it did not induce a rapid increase in type X collagen mRNA. In contrast, BMP added to serum-free medium induced a three- to fourfold increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required. Addition of thyroid hormone to serum-free medium was required for maximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chondrocytes, which do not undergo hypertrophy during embryonic development. These results indicate that ascorbate + BMP in serum-free culture induces rapid chondrocyte maturation of prehypertrophic chondrocytes. The mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction. J. Cell. Biochem. 66:394-403, 1997. © 1997 Wiley-Liss, Inc.
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  • 97
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    Carboxyl terminus of mitosin is sufficient to confer spindle pole localization (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 441-449 
    Details
    ISSN: 0730-2312
    Keywords: mitosin ; CENP-F ; spindle pole ; kinetochore ; centromere ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mitosin is a nuclear protein of 3,113 amino acids which has been shown to associate with the mitotic apparatus, especially the kinetochore, during mitosis. In this paper we further confirmed its association with the spindle poles in normal monkey kidney CV1 cells by indirect immunofluorescence microscopy. When the carboxyl portion of mitosin containing amino acids 2,094-3,113 (named mitosin-pTN) was stably expressed in rat fibroblast Rat2 cells using a tetracycline-inducible system, strong spindle pole association was observed in addition to expected centromere localization. The same results were achieved in Chinese hamster ovary (CHO) cells. On the other hand, mitosin-pTC containing amino acids 2,756-3,113 was not targeted to spindle poles. Use of the FLAG epitope [Hopp et al., 1988] genetically fused to each amino terminus of these mutants eliminated possible artifacts due to antibody cross-reaction, since the spindle pole localization of wild-type mitosin was confirmed with a FLAG-tagged mutant by an antibody (anti-FLAG M2 monoclonal antibody) irrelevant to antibodies to mitosin. Our data also suggested a possible interaction of mitosin with the spindle microtubules. Interaction of mitosin with the major parts of the mitotic apparatus further implies an important role in mitosis. J. Cell. Biochem. 66:441-449, 1997. © 1997 Wiley-Liss, Inc.
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  • 98
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    Evidence for inhibition of MyEF-2 binding to MBP promoter by MEF-1/Pur α (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 524-531 
    Details
    ISSN: 0730-2312
    Keywords: MBP ; brain development ; transcription ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Myelin basic protein (MBP) is a major component of the myelin sheath whose production is developmentally controlled during myelinogenesis. Earlier studies have indicated that programmed expression of the MBP gene is regulated at the level of transcription. Evidently, the MB1 regulatory motif located between nucleotides -14 to -50 plays an important role in transcription of the MBP promoter in both in vitro and in vivo systems. The MB1 element contains binding sites for the activator protein MEF-1/Pur α and the repressor protein MyEF-2. In this study we use bandshift assays with purified MEF-1/Pur α and MyEF-2 and demonstrate that binding of MyEF-2 to its target sequence is inhibited by MEF-1/Pur α. Under similar conditions, MyEF-2 enhances the association of MEF-1/Pur α with MB1 DNA. MEF-1/Pur α binds to MB1 in mono- and dimeric forms. Inclusion of MyEF-2 in the binding reaction increases the dimeric association of MEF-1/Pur α with the MB1 sequence. The use of MEF-1/Pur α variants in the bandshift assay suggests that two distinct regions of this protein may be involved in its binding to the MB1 sequences, and its ability to block MyEF-2 interaction with the MB1 sequence. Based on previous studies on the programmed expression of MEF-1/Pur α and MyEF-2 during myelination and the current findings on their interplay for binding to the MB1 motif, a model is proposed for their involvement in transcriptional regulation of the MBP gene during the course of brain development. J. Cell. Biochem. 66:524-531, 1997. © 1997 Wiley-Liss, Inc.
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  • 99
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    Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 33-42 
    Details
    ISSN: 0730-2312
    Keywords: apoptosis ; cysteine proteases ; CPP32 ; Mch2 ; Mch3 ; Mch4 ; Mch5 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: So far nine human aspartate-specific cysteine proteases (ASCPs) have been identified and cloned in our lab and others. Their sequence and structural homology to the nematode Ced-3 implicated them in the cell death pathway of mammalian cells. Recent evidence suggests that ASCPs initiate apoptosis by acting at or near the cell death effector level. However, it is not clear whether the activity of one or several of these enzymes is necessary for execution of apoptosis. In addition, it is not yet clear how the proenzymes of ASCPs are activated or what triggers their activation. Execution of apoptosis in higher eukaryotes is apparently more complicated than in nematodes. It is most likely that in mammalian cells this process involves the coordinated action of multiple ASCPs and multiple redundant proteolytic pathways. J. Cell Biochem. 64:33-42. © 1997 Wiley-Liss, Inc.
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  • 100
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    Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 295-312 
    Details
    ISSN: 0730-2312
    Keywords: osteoblast ; glucocorticoids ; hydroxyapatite ; osteoprogenitor ; bone marrow ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM β-glycerophosphate (βGP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM βGP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number or APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes. J. Cell. Biochem. 64:295-312. © 1997 Wiley-Liss, Inc.
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