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  • 1995-1999  (666)
  • 1980-1984  (921)
  • 1975-1979  (751)
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  • 1
    ISSN: 1437-160X
    Keywords: Rheumatoid pannus ; Adhesion molecules ; β1 integrins ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To investigate the mechanism of synovial pannus formation in rheumatoid arthritis, immunohistochemical and immunoelectron microscopic studies with monoclonal antibodies against the adhesion molecules, CD54 (ICAM-1), CD 11a (LFA-1), CDw49a (VLA-1), CDw49b (VLA-2), CDw49c (VLA-3), Cdw49d (VLA-4) and CDw49e (VLA-5), were carried out to determine the pattern of distribution of these molecules at the rheumatoid synovial cartilage junction. Treatment with anti-ICAM-1 resulted in membrane staining of most of the macrophages and fibroblasts infiltrating the synovial tissue and bordering the pannus-cartilage junction, suggesting the possibility that ICAM-1 may function to facilitate the adhesion of synovial type A cells bearing ICAM-1 to type B cells of the pannus. ICAM-1-positive macrophages and fibroblasts were often found to be in contact with lymphoid cells, suggesting also that a cellular immune reaction occurs in the formation of the pannus. In addition, VLA-3, VLA-4 and, particularly, VLA-5 were the predominantβ1 integrins expressed by rheumatoid synovial pannus. Since these three integrins all function as fibronectin receptors, it is possible that the fibronectin-rich environment of the rheumatoid cartilage surface effectively traps pannus cells expressing high levels of these molecules. The VLA-5 molecule was found in a pericellular and interterritorial matrix distribution in the present study, strongly suggesting that a recepfor-ligand interaction between VLA-5 and cartilage matrix may occur at the early stage of pannus formation. Furthermore, an increase inβ1 integrin may be necessary for the growth of the pannus and also for the upregulation of the VLA molecules, leading secondarily to increased attachment.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Neurosurgical review 19 (1996), S. 39-42 
    ISSN: 1437-2320
    Keywords: Electron microscopy ; immunohistochemistry ; meningeal melanocytoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A case of cervical juxtamedullary meningeal melanocytoma is presented along with results of careful neuroradiological and pathological examinations. The authors review the previous literature and discuss the problems related to the intriguing diagnosis of this very rare entity. CT and MRI show variable appearances due to different degrees of melanization and do not reliably allow us to distinguish meningeal melanocytomas from other pigmented tumors. Pathological examination of the tumor is critical for diagnosis, but is absolutely must comprise electron microscopy and immunohistochemistry. Immunohistochemical and ultrastructural criteria are thus proposed to distinguish meningeal melanocytoma from pigmented meningioma or schwannoma and malignant melanoma (primary or secondary).
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1433-0423
    Keywords: Schlüsselwörter Makulaforamen ; Epiretinale Membran ; Elektronenmikroskopie ; Vitrektomie ; Pathogenese ; Key words Macular hole ; Epiretinal membrane ; Electron microscopy ; Vitrectomy ; Pathogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Anteroposterior and tangential traction on the central retina is an important factor in the pathogenesis of idiopathic macular hole formation. Histological studies have shown that macular holes of different stages can be associated with epiretinal membranes. Such membranes can be removed during surgery for macular holes. We investigated such tissue samples of 11 patients with macular holes in stages II – IV. Light microscopially, the tissue consisted of a thin collagen layer mostly covered by a thin layer of cells. Ultrastructural analysis revealed glial cells and macrophages as cellular components. The collagen can be ascribed to vitreous, inner limiting membrane and newly formed collagen. According to the morphological findings a multilayered tissue structure can be assumed. Macrophages were found on the retinal side of the inner limiting membrane and at the vitreal side of the tissue. Therefore, the macrophages probably originate from the retina as well as from the vitreous as so-called resident hyalocytes. Glial cells covered the inner limiting membrane forming pericellular collagen to which outer vitreous collagen fibrils can be attached. The multilayered membrane structure might possibly be the cause for only partial laminar surgical extraction so that contractile or potentially proliferative tissue residues might be one of the reasons for surgical failures after incomplete membrane peeling.
    Notes: A.-p. und tangentiale Traktionen an der zentralen Netzhaut spielen für die Entstehung idiopathischer Makulaforamina eine wichtige Rolle. Durch histologische Untersuchungen ließen sich bei Makulaforamina unterschiedlicher Stadien epiretinale Membranen nachweisen, die für die tangentiale Traktion auf die Foramenränder verantwortlich gemacht werden. Von 11 Patienten konnten wir derartige, von uns chirurgisch entfernte Membranen licht- und elektronenmikroskopisch untersuchen. Die Membanen bestehen lichtmikroskopisch aus einem azellulären Kollagenband mit einem schmalen, teilweise nur einschichtigen Zellbesatz. Elektronenmikroskopisch lassen sich als zelluläre Komponenten Gliazellen und Makrophagen differenzieren. Die Kollagenanteile sind Glaskörperkollagen, neugebildetem Kollagen und der Lamina limitans interna zuzuordnen. Aus den Befunden läßt sich eine Schichtung der Membranen rekonstruieren mit sublaminären Makrophagen, der Lamina limitans interna und epilaminären Gliazellen und vereinzelten Makrophagen, die am ehesten residenten Hyalozyten entsprechen. Auf der vitrealen Seite können auch Verbindungen der Membran zum äußeren Glaskörperkollagen bestehen. Die Membranen zeigen Charakteristika, die in ihrem Aufbau epiretinalen Proliferationsmembranen entsprechen. Die topographische Schichtung der Membranen könnte zur Folge haben, daß chirurgisch teilweise nur Lamellen und nicht die gesamte Membran abgeschält werden. Kontraktions- oder auch proliferationsfähige Membranreste können dann einer der Faktoren für operative Mißerfolge sein.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Pediatric surgery international 12 (1996), S. 49-53 
    ISSN: 1437-9813
    Keywords: Key words Silicone ; Silicon tubing ; Ivac 560 pump ; Energy-dispersive X-ray analysis (EDXA) ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Migration of particulate matter from plastic tubing and solid plastic implants has been documented in a number of studies, including some with the use of cardiac bypass, haemodialysis, and pump-assisted intravenous infusions. In order to ascertain whether silicone embolisation occurs when children have an Ivac 560 pump-assisted IV infusion, we passed 180 ml of pumped fluid through a microfilter and compared the scanning electron micrographs of those filters with unused filters and with others through which a similar volume had been passed without using the pump. The particles on the filters were analysed for their elemental content using energy-dispersive X-ray analysis. In addition, the appearance of the silicone tubing used in the pump over 3 and 72 h was assessed and compared to that of flow-only and unused tubing. More particles were found on the microfilter when fluid had been delivered via the pump than on those through which non-pumped fluid had passed or that were unused. Elemental silicon-containing particles were only found on the filter when a pump had been attached to the IV line. The flow-only and unused tubing were found to have adherent particles on the inner surface that were not seen once the tubing had been used for 3 h in the Ivac 560 pump. Also, after 72 h use, the silicone tubing had a deformed inner layer. The clinical significance of these findings is yet to be determined, but it does appear that silicone embolisation occurs during pump-assisted infusions in children.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Pediatric surgery international 12 (1996), S. 49-53 
    ISSN: 1437-9813
    Keywords: Silicone ; Silicon tubing ; Ivac 560 pump ; Energy-dispersive X-ray analysis (EDXA) ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Migration of particulate matter from plastic tubing and solid plastic implants has been documented in a number of studies, including some with the use of cardiac bypass, haemodialysis, and pump-assisted intravenous infusions. In order to ascertain whether silicone embolisation occurs when children have an Ivac 560 pump-assisted IV infusion, we passed 180 ml of pumped fluid through a microfilter and compared the scanning electron micrographs of those filters with unused filters and with others through which a similar volume had been passed without using the pump. The particles on the filters were analysed for their elemental content using energy-dispersive X-ray analysis. In addition, the appearance of the silicone tubing used in the pump over 3 and 72 h was assessed and compared to that of flow-only and unused tubing. More particles were found on the microfilter when fluid had been delivered via the pump than on those through which nonpumped fluid had passed or that were unused. Elemental silicon-containing particles were only found on the filter when a pump had been attached to the IV line. The flow-only and unused tubing were found to have adherent particles on the inner surface that were not seen once the tubing had been used for 3 h in the Ivac 560 pump. Also, after 72 h use, the silicone tubing had a deformed inner layer. The clinical significance of these findings is yet to be determined, but it does appear that silicone embolisation occurs during pump-assisted infusions in children.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-2307
    Keywords: Papillary adenoma of type II pneumocytes ; Immunohistochemistry ; Electron microscopy ; Morphometry ; Multivariate cluster analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Papillary adenoma of type II pneumocytes is a rare tumour. It is considered to be a benign neoplasm and is derived from immature cells in the bronchioloalveolar epithelium, however, its biological nature has not been elucidated. We report a case of an adenomatous tumour; a papillary adenoma of type II pneumocytes, which we regard as possessing malignant potential. Light microscopically, as well circumscribed, papillary tumour of predominantly cuboidal cells resembling type II pneumocytes was found, but Clara type and ciliated cells were also present. Immunohistochemically, the tumour cells reacted positively with antibodies to surfactant apoproteins (A, B), carcinoembryonic antigen, cytochrome P-450 1A1-2 and 2B1-2. Ultrastructurally, many osmiophilic lamellar bodies and electron-dense granules were demonstrated. Semi-serial sections revealed signs of transbronchial dissemination and vascular invasion. Morphometry using 12-dimensional cluster analysis disclosed features of the tumour cells which resembled those of pneumocyte type II adenocarcinoma. These findings suggest that the present case has some malignant characteristics and originates from immature bronchiolar or alveolar cells, with a potential to develop into both type II pneumocyte and Clara cell type adenocarcinomas.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1434-0879
    Keywords: Interstitial stroma ; Carcinogenesis ; Bladder epithelium ; Neovascularization ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract This study describes ultrastructural alterations of the interstitial stroma in the rat bladder epithelium during N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis using scanning (SEM) and transmission (TEM) electron microscopy with NaOH treatment. The results obtained with SEM demonstrated the occurrence and development of stroma protrusions which exhibited pipe-like structures in the rat bladder epithelium following administration of BBN. Number and size of blood vessel sections also gradually increased both in the stroma and within the layer of the proliferated epithelial cells as examined by light microscopy (LM) and TEM. In this study stroma alterations were not only observed in malignant lesions of rat bladder, but hyperplastic lesions were also accompanied by stroma alterations. It is suggested that: (1) the interstitial stroma of the rat bladder epithelium may exhibit pathological changes in structure and these changes may correlate with the progression of epithelial cell proliferation following administration of BBN and (2) one of the most important alterations in the stroma is the occurrence of neovascularization, which may induce structural modification of the stroma in the processes of bladder tumor growth and development.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 193 (1996), S. 331-339 
    ISSN: 1432-0568
    Keywords: Synapse ; Cell migration ; Olfactory neurons ; Electron microscopy ; Vomeronasal organ
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Rat neonate vomeronasal organs were transplanted into the parietal cortex of littermates to examine their survival and the behavior of axon growth into the surrounding host brain parenchyma. After survival times of 10–100 days the brains were processed for ultrastructural examination. The transplanted vomeronasal organs (VNO) formed several vesicles lined with a sensory epithelium. From these sensory epithelia, VNO neurons leave the epithelium and enter the host brain. Transplant neurons grew axons that fasciculated into bundles surrounded by sheath cell processes and formed one or more fiber plexuses containing distinct globose or spherical-shaped glomeralar-like structures. The glomeruli consisted of nerve terminals between which existed asymmetric synaptic contacts. Rarely did we observe clear reciprocal synapses. The glomeruli also contained terminals that showed signs of degeneration, such as increased density of the terminals, clumping of mitochondria and multivesicular bodies. The glomeruli were not partitioned or subdivided by glial septa; however, glial profiles were interspersed among the sensory terminals. Transplant glomeruli also lacked periglomerular cells and had no definitive glial envelope. These results suggest that glomerular formation is not dependent on dendrite contribution of second order neurons or glial support, but rather on a complementary population of receptor neurons.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-0568
    Keywords: Thalamus ; Somatosensory system ; Synaptogenesis ; Nervous system ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Electron microscopy has been employed to analyze the normal maturationai sequence that characterizes the postnatal development of synaptic circuits in the ventrobasal (VB) and reticular (Rt) thalamic nuclei of rats at different ages (from birth to the end of the third postnatal week). Throughout the first postnatal week, similar signs of immaturity are observed in both nuclei, mainly consisting in scarcity of cytoplasmic organelles, presence of wide extracellular spaces, and absence of myelinated fibers. Several synaptic terminals are however present from birth, thus indicating that some of the afferents have already reached and contacted their thalamic target during embryonic life. Most of the terminals are small and contain only a few round, clear vesicles, and therefore their cytological features do not allow the identification of their origin. In particular, in both nuclei, terminals with flat vesicles and symmetric specialization are only rarely observed, and in VB the ascending terminals are not distinguishable from terminals of other sources as they are in adults. During the second postnatal week, progressive maturationai changes in VB and Rt lead to neurons having well-developed cytoplasmic organelles and to an elaborate neuropil containing myelinated fibers and synaptic terminals that are morphologically heterogeneous and resemble the adult ones. The permanence of growth cone-like profiles and of numerous somatic and dendritic protrusions, often contacted by synaptic terminals, indicates that a certain degree of reorganization is still taking place in both nuclei. By the end of the third postnatal week the synaptic organization of VB and Rt is indistinguishable from that observed in adults. This ultrastructural study shows that the appearance of the neuropil of VB and Rt and the morphological complexity of the synaptic arrangements characteristic of the adult rat are not present in neonates, but are gradually acquired during the first three postnatal weeks, and that they result from progressive modifications in circuit organization involving both pre- and postsynaptic elements.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0533
    Keywords: Key words Fukuyama-type congential muscular ; dystrophy ; Fetus ; Cerebrocortical pathology ; Breaches in glia limitans ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A light and electron microscopic study of the brain of an 18-week fetus with a prenatal genetic diagnosis of Fukuyama-type congenital muscular dystrophy revealed a widespread mantle of abnormal neurogliomesenchymal tissue that covered a dysplastic cerebral cortex. In this area alone, the glia limitans that adjoined the abnormal mantle via one or two layers of basal lamina had frequent breaches, through which neuroglial elements extruded. In the most severely affected cortical region, which had only a rudimentary and fragmentary glia limitans, the majority of cortical neurons had migrated into the neurogliomesenchymal tissue. The massive overmigrated neurons still maintained a somewhat columnar arrangement, and the marked dysplasia abruptly shifted to a neurogliomesenchymal tissue-free normal cortical structure with an intact glia limitans, thus indicating essentially vertical overmigration of neurons without significant tangential migration of them. Together the above findings imply that breaches in the glia limitans may be the primary cause of the micropolygyria seen in this genetic disorder.
    Type of Medium: Electronic Resource
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  • 11
    ISSN: 1432-0533
    Keywords: Key words Central neurocytoma ; Glial fibrillary acidic protein ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have identified two central neurocytomas which contained cells co-expressing glial fibrillary acidic protein and synaptophysin defined by double-label immunostaining. Dual-positive cells were mostly polygonal in shape and with a morphological appearence similar to that of reactive astrocytes. This distinct morphology could be used to distinguish cells expressing glial fibrillary acidic protein from cells with round and clear cytoplasm which did not express glial fibrillary acidic protein and which composed the majority of the tumor. Samples containing polygonal cells were selected for electron microscopy from toluidine blue-stained semithin sections. Ultrastructural findings were similar in both neurocytomas, with both being composed predominantly of round cells with clear cytoplasm corresponding to the clear cells identified by light microscopy. Dense-core vesicles and clear vesicles were frequently observed in the cell processes. Apart from these clear cells, polygonal cells with electron-dense cytoplasm were noted. Paralleling the results of double immunostaining, these polygonal cells contained both dense-core vesicles and intermediate, presumably glial filaments. Microtubules and lipofuscin granules were also observed. These results suggest that cells expressing glial fibrillary acidic protein in central neurocytoma include tumor cells with both neuronal and glial characteristics.
    Type of Medium: Electronic Resource
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 193 (1996), S. 259-269 
    ISSN: 1432-0568
    Keywords: Stomach ; Parietal cell lineage ; Heterogeneity ; Electron microscopy ; Cattle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The origin and differentiation of the oxyntic cell lineage during physiological cell renewal was investigated by light and electron microscopy in the abomasal mucosa of adult cattle. The morphologically heterogeneous oxyntic cell population exhibits various developmental subtypes depending on the position within the oxyntic unit. Pre-oxyntic cells of the isthmus and neck represent the immature precursors. Though heterogeneous with respect to the degree of canalicular and tubulovesicular membrane development, they all contain secretory granules resembling those of either isthmus cells, immature surface mucous cells, neck cells or young chief cells. A secretory granule-free stem cell is not present in the bovine. Downward to the gland base genesis of canalicular as well as tubulovesicular membranes is gradually completed; thus pre-oxyntic cells give rise to mature oxyntic cells. Older degenerative oxyntic cells, primarily located within the gland bottom, are characterized by progressive involution of canalicular and tubulovesicular membranes. Towards the pit, differentiation of pre-oxyntic cells is associated with atypical and incomplete development of canaliculi and tubulovesicles. In consequence, these superficial oxyntic cells have a reduced secretory capacity from a morphological point of view.
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Archives of dermatological research 288 (1996), S. 91-97 
    ISSN: 1432-069X
    Keywords: Key words Hemidesmosomes ; Basement membrane ; Fetal skin ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Hemidesmosomes are junctional complexes involved in the attachment of epidermal basal keratinocytes to the basement membrane. To try to understand better the sequence of events in the morphogenesis of hemidesmosomes, we undertook an ultrastructural analysis of hemidesmosome formation in fetal and neonatal digit skin. Hemidesmosomes, defined as membrane-associated densities or plaques, were counted and scored for three morphological characteristics: (1) the presence of a sub-basal dense plate, (2) association with anchoring filaments within the lamina lucida and (3) contacts with intermediate filaments. No hemidesmosomes were evident at 7 weeks’ gestational age. Between 9 and 15 weeks the number of hemidesmosomes increased by about fourfold (from 20.6 ± 3.8 (SD) to 95.5 ± 8.4 per 40 μm of basal cell plasma membrane; P 〈 0.01). The association of hemidesmosomes with intermediate filaments and anchoring filaments also increased after 15 weeks ( P 〈 0.05). Early attachment plaques first appeared as triangular focal densities on the basal plasma membrane with the appearance of sub-basal dense plates, which later became both larger and more electron dense. By 15 weeks, an inner plaque could be distinguished from the outer plaque, which coincided with a closer association with intermediate filaments. Hemidesmosomes appeared fully developed by 15 weeks’ gestation. This study illustrates the structural relationship of hemidesmosomes to both intra- and extracellular filaments, suggesting close functional interactions. The complexity of the hemidesmosome plaque is also revealed early during development.
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  • 14
    ISSN: 1432-1173
    Keywords: Schlüsselwörter Papillomatosis confluens et reticularis ; Minocyclin ; Tetracycline ; Elektronenmikroskopie ; Key words Confluent and reticulated papillomatosis ; Minocycline ; Tetracycline ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Confluent and reticulated papillomatosis (CRP) is a rare dermatosis of unknown aetiology. Recent electron microscopic studies suggest that CRP is a disorder of keratinisation. In our case we could not confirm the previously reported ultrastructural findings. CRP is generally resistant to therapy. We treated a 19-year-old patient with typical CRP with oral minocycline. Within a few weeks the eruption resolved completely. A mild relapse 7 months later responded promptly to a repeated course of minocycline. Twelve months after discontinuatin of therapy there is no evidence of recurrence. In CRP minocycline should be preferred to systemic retinoid therapy because of its minor side effects.
    Notes: Zusammenfassung Die Papillomatosis confluens et reticularis (PCR) ist eine seltene Erkrankung unklarer Ätiopathogenese. Neuere elektronenmikroskopische Untersuchungen stützen die Vermutung, daß eine Keratinisierungsstörung vorliegt. Anhand unseres Falles konnten wir diese Befunde allerdings nicht bestätigen. Die PCR ist ausgesprochen therapieresistent. Wir behandelten einen 19jährigen Patienten mit klinisch und histologisch typischer PCR systemisch mit Minocyclin. Innerhalb weniger Wochen heilten die Effloreszenzen vollständig ab. Ein 7 Monate später auftretendes leichtes Rezidiv konnte durch einen zweiten Behandlungszyklus schnell und erfolgreich behandelt werden. Der Patient ist jetzt bereits 12 Monate nach Absetzen der Therapie erscheinungsfrei. Zur Behandlung der PCR sollte Minocyclin wegen seiner geringeren Nebenwirkungen systemischen Retinoiden vorgezogen werden.
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  • 15
    ISSN: 1432-1106
    Keywords: GluR1 ; GABA ; Electron microscopy ; Cerebral neocortex ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Specimens of human cerebral neocortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the glutamate receptor subunit GluR1 and gamma-aminobutyric acid (GABA). Many GluR1-positive pyramidal neurons and fewer GluR1-positive nonpyramidal neurons were present in the cortex. Non-pyramidal neurons were more heavily labelled for GluR1 than pyramidal neurons. Most GABAergic neurons were labelled for GluR1. The white matter was unstained, except for occasional labelled neurons. This pattern of GluR1 immunostaining is similar to that in rat cerebral cortex, but is different from that in the hippocampus and amygdala, where large numbers of pyramidal or projection neurons, but few non-pyramidal or GABAergic neurons, were labelled for GluR1.
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  • 16
    ISSN: 1432-1106
    Keywords: Glutamate receptor ; Cerebral cortex ; White matter ; Electron microscopy ; Development ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The distribution of the [3H]alpha-amino-3-hydroxy-5-methylisoxzalepropionic acid (AMPA) receptor subunit GluR4 was studied in frontal, parietal and temporal cerebral cortex, subcortical white matter and corpus callosum of neonatal, immature and mature rats. In 1-to 2-day-old rats, a few oligodendrocyte progenitors and amoeboid microglia in the supraventricular part of the corpus callosum were immunolabelled for GluR4. At 7 to 10 days, the number of amoeboid microglia and oligodendrocyte progenitors in white matter increased; many neurons in cortex, including pyramidal neurons, were also moderately labelled for GluR4. The pattern of GluR4 immunostaining in 14-day-old rats was different from that in 7-to 10-day-old rats, but similar to the adult, in that there was no immunoreactivity in microglia and oligodendrocyte progenitors in subcortical white matter. A proportion of non-pyramidal neurons in cortex were moderately labelled, while some pyramidal neurons were lightly labelled. A population of small glial cells with features of oligodendrocyte progenitors were densely labelled in cortex.
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    European journal of pediatrics 155 (1996), S. 561-567 
    ISSN: 1432-1076
    Keywords: Key words Glycogen deficiency ; Glycogen synthase ; Ketotic ; hypoglycaemia ; Corn (maize) ; starch ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Three children from two German families are described and the observations compared with the previously published three families comprising eight patients. The two index cases presented with morning fatigue, had ketotic hypoglycaemia when fasting which rapidly disappeared after eating, and hepatic glycogen deficiency and absent or very low hepatic glycogen synthase activity. Metabolic profiles comprising glucose, lactate, alanine, and ketones in blood were typical for hepatic glycogen synthase deficiency. Symptoms were rapidly relieved and chemical signs corrected by introducing frequent protein-rich meals and night-time feedings of suspensions of uncooked corn (maize) starch. The discovery of oligosymptomatic and asymptomatic siblings suggests that there are more persons with undiagnosed hepatic glycogen synthase deficiency. Conclusion Liver glycogen synthase deficiency is likely to be more common than is believed today. It should be sought in children who, before the first meal of the day, present with drowsiness, lack of attention, pallor, uncoordinated eye movements, disorientation or convulsions and who have hypoglycaemia and acetone in urine.
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    European journal of pediatrics 155 (1996), S. 561-567 
    ISSN: 1432-1076
    Keywords: Glycogen deficiency ; Glycogen synthase ; Ketotic hypoglycaemia ; Corn (maize) starch ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Three children from two German families are described and the observations compared with the previously published three families comprising eight patients. The two index cases presented with morning fatigue, had ketotic hypoglycaemia when fasting which rapidly disappeared after eating, and hepatic glycogen deficiency and absent or very low hepatic glycogen synthase activity. Metabolic profiles comprising glucose, lactate, alanine, and ketones in blood were typical for hepatic glycogen synthase deficiency. Symptoms were rapidly relieved and chemical signs corrected by introducing frequent protein-rich meals and night-time feedings of suspensions of uncooked corn (maize) starch. The discovery of oligosymptomatic and asymptomatic siblings suggests that there are more persons with undiagnosed hepatic glycogen synthase deficiency.
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  • 19
    ISSN: 1432-1017
    Keywords: Key words Gallstone ; Cholesterol monohydrate crystals ; Phase separation ; Light scattering ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Phospholipid/cholesterol vesicles were solu-bilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Above 30 mol% cholesterol (Ch) in the lipid vesicles several remarkable changes of the solubilization process were observed. (i) Two modes of solubilization: The effective detergent to lipid ratio Rc(M) for the formation of mixed micelles decreased from Rc(M) = 43 ± 3 at low lipid concentrations, [L]≤ 0.15 mm, to Rc(M) = 2.4 ± 0.3 above [L] = 0.5 mm (40 mol% Ch, T = 20 °C). (ii) At subsolubilizing CHAPS concentrations, filamentous and helical microstructures were formed, similar to those which were observed in native and model bile. (iii) The number of observed fibers was about two orders of magnitude higher in the presence of the negatively charged lipids phosphatidylglycerol (PG) and phosphatidic acid (PA) compared to the zwitterionic phosphatidylcholine (PC). Fiber formation began after 16–18 h using PG and PA compared to 3–4 days in the presence of PC. Screening of the charged lipids by NaCl effectively reduced the formation of fibers. Assuming binding of Na+ to the charged lipid aggregates, an intrinsic binding constant Kint = 0.6 M–1 was determined by applying the Gouy-Chapman theory. After the addition of CHAPS to PG/Ch vesicles, a fast initial solubilization of the vesicles (〈1 min) to mixed micelles (rh = 2.3 ± 0.2 nm) and small vesicles (rh = 23 ± 1 nm) was observed, followed by an intermediate period of 2 h, after which the formation of fibers occurred (〉15 h). The microstructures are visualized by darkfield and electron microscopy. The method of vesicle solubilization is compared to the dilution of concentrated micellar solutions, which is usually applied to model bile systems.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Biology and fertility of soils 21 (1996), S. 293-302 
    ISSN: 1432-0789
    Keywords: Key words Ammonium excretion ; Azorhizobium caulinodans ; Auxine ; 2 ; 4-Dichlor-phenoxy-acetic acid ; Nitrogen fixation ; Paranodulation ; Rice ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Rice seedlings developed nodule-like tumors (para-nodules) along primary and secondary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Histologically, these tumors appeared as cancerous out-grown lateral-root primordes and were thus comparable with stem nodules of the legume Sesbania rostrata. Azorhizobium caulinodans (a diazotroph known as a specific endophyte of Sesbania rostrata) was introduced and became established inside rice para-nodules and in root tissues around tumor bases. The infection with A. caulinodans followed a typical “crack-entry” invasion at places where para-nodule tumors had emerged through the root cortex and epidermis. The bacteria settled with high cell densities in intercellular spaces of the induced tumors and betwen root cortical cells. Infection of plant cells took place both in the epidermis and in cortical tissue. Intracellularly established A. caulinodans was found inside the cytoplasm, surrounded by membrane-like structures. N2 fixation by tumor-inhabiting Azorhizobium sp. was increased at low O2 tensions (1.5–3 kPa) compared with an untreated control. Only a little activity remained at O2 tensions of 5 kPa and above. The present results confirm that root-tumor induction offers a suitable method of establishing diazotrophs endophytically in the roots of gramineous crops.
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  • 21
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    Biology and fertility of soils 21 (1996), S. 293-302 
    ISSN: 1432-0789
    Keywords: Ammonium excretion ; Azorhizobium caulinodans ; Auxine 2.4-Dichlor-phenoxy-acetic acid ; Nitrogen fixation ; Paranodulation ; Rice ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Rice seedlings developed nodule-like tumors (para-nodules) along primary and secondary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Histologically, these tumors appeared as cancerous out-grown lateral-root primordes and were thus comparable with stem nodules of the legume Sesbania rostrata. Azorhizobium caulinodans (a diazotroph known as a specific endophyte of Sesbania rostrata) was introduced and became established inside rice para-nodules and in root tissues around tumor bases. The infection with A. caulinodans followed a typical “crack-entry” invasion at places where paranodule tumors had emerged through the root cortex and epidermis. The bacteria settled with high cell densities in intercellular spaces of the induced tumors and between root cortical cells. Infection of plant cells took place both in the epidermis and in cortical tissue. Intracellularly established A. caulinodans was found inside the cytoplasm, surrounded by membrane-like structures. N2 fixation by tumor-inhabiting Azorhizobium sp. was increased at low O2 tensions (1.5–3 kPa) compared with an untreated control. Only a little activity remained at O2 tensions of 5 kPa and above. The present results confirm that root-tumor induction offers a suitable method of establishing diazotrophs endophytically in the roots of gramineous crops.
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  • 22
    ISSN: 1432-1076
    Keywords: Key words Rhizomelic ; chondrodysplasia punctata ; Dihydroxyacetonephosphate-acyl-transferase ; Electron microscopy ; Peroxisomes ; Morphometry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Rhizomelic chondrodysplasia punctata (RCDP) is clinically characterized by symmetrical shortening of the proximal limbs, contractures of joints, a characteristic dysmorphic face, and cataracts. In the classical form an impairment of several peroxisomal functions and enzymes (plasmalogen synthesis, phytanic acid oxidation, 3-oxoacyl-CoA thiolase) has been repeatedly shown. Recently a variant involving only the peroxisomal dihydroxyacetonephosphate acyltransferase (DHAP-AT) has been described. We present a patient with isolated DHAP-AT deficiency and all clinical, radiological, and pathological features of classical RCDP. For the first time, microscopy and immunocytochemistry of hepatocytes could be performed. Conclusion In contrast to studies on classical rhizomelic chondrodysplasia punctata which have shown enlarged peroxisomes in numbers varying from hepatocyte to hepatocyte, the peroxisomes in our patient seem to be normal in size, number and shape.
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  • 23
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    Archives of microbiology 166 (1996), S. 357-360 
    ISSN: 1432-072X
    Keywords: Key wordsEscherichia coli ; Capsule ; Serotype ; Edema disease ; Electron microscopy ; Cell adhesion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The fine structure of the cell surface of seven enterotoxemic Escherichia coli (ETEEC) O139:K12 strains isolated from piglets with edema disease were examined electron microscopically using both the negative-staining method and the freeze-substitution fixation method. Densely packed, fine fibers were observed; they consisted of a capsule layer approximately 25 nm thick around the cell surfaces of strains 107/86, IW-2, ED-3, ED-43, and ED-61, all of which have a capacity to adhere strongly to HEp-2 cells. In contrast, no such structure was observed on the surface of strains RK-O139 or ED-1, both of which adhere only weakly to HEp-2 cells. These results suggest that the capsule structure might be associated with the ability to adhere to HEp-2 cells and, as a result, also potentially play some role in ETEEC infection.
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  • 24
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    Bioscience reports 16 (1996), S. 159-187 
    ISSN: 1573-4935
    Keywords: Electron microscopy ; photosystem II ; thylakoid membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract This review covers the recent progress in the elucidation of the structure of photosystem II (PSII). Because much of the structural information for this membrane protein complex has been revealed by electron microscopy (EM), the review will also consider the specific technical and interpretation problems that arise with EM where they are of particular relevance to the structural data. Most recent reviews of photosystem II structure have concentrated on molecular studies of the PSII genes and on the likely roles of the subunits that they encode or they were mainly concerned with the biophysical data and fast absorption spectroscopy largely relating to electron transfer in various purified PSII preparations. In this review, we will focus on the approaches to the three-dimensional architecture of the complex and the lipid bilayer in which it is located (the thylakoid membrane) with special emphasis placed upon electron microscopical studies of PSII-containing thylakoid membranes. There are a few reports of 3D crystals of PSII and of associated X-ray diffraction measurements and although little structural information has so far been obtained from such studies (because of the lack of 3D crystals of sufficient quality), the prospects for such studies are also assessed.
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  • 25
    ISSN: 1432-2307
    Keywords: Acinar cell carcinoma ; Fetal pancreas ; Electron microscopy ; Fibrillary inclusion ; Rough endoplasmic reticulum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report a case of pancreatic acinar cell carcinoma which contained a large number of pleomorphic inclusions with fibrillary internal structures and mature zymogen granules. To clarify the significance of fibrillary inclusions in the differentiation of acinar cells of the pancreas, we further investigated fetal pancreases (gestational weeks 16, 17, 19, 20 and 28). We found two types of inclusions: type A, corresponding to fibrillary inclusion of neoplastic acinar cells, was observed only in a 19-week fetus; type B showed a homogeneous density similar to that of zymogen granules. Type B was observed in all the fetuses after the 17th gestational week. Although the type A inclusion might be generated throught a different mechanism than the type B inclusion, the appearance of a large number of fibrillary inclusions in neoplastic acinar cells may represent a transient form of zymogen granule.
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  • 26
    ISSN: 1432-2307
    Keywords: Alveolar adenoma ; Lung neoplasms ; Flow cytometry ; Electron microscopy ; Proliferating cell nuclear antigen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Alveolar adenoma of the lung is a poorly characterized, uncommon pulmonary lesion with proliferation of alveolar epithelium and septal mesenchyme. We describe the electron microscopy, immunohistochemistry and DNA flow cytometry in a case of alveolar adenoma in a 55-year-old woman. Alveolar adenoma appears to be a distinct benign neoplasm of the alveolar structures. Our findings further suggest that it is not a precursor of bronchioloalveolar carcinoma or other type II pneumocyte lesions of presumed malignant potential.
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  • 27
    ISSN: 1432-0878
    Keywords: Key words: Electrocyte ; Intermediate filaments ; Desmin ; Cytoskeleton ; Electron microscopy ; Electrophorus electricus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Desmin protein is an abundant constituent of the intermediate filaments in the electrocytes of the electric organ of the electric eel Electrophorus electricus. Polyclonal antibodies were raised against purified desmin from the electric organ and used for immunolabeling of the protein in reconstituted filaments. In thick sections of the main electric organ that has been stained with fluorescein-labeled desmin-specific antibodies, light microscope revealed a diffuse meshwork of desmin filaments dispersed in the cytoplasm of electrocytes. In the region under the membrane, the immunostaining was slightly more intense than elsewhere. The meshwork of intermediate filaments composed of desmin was examined by electron microscopy of the main electric organ. Immuno-gold labeling demonstrated a widespread meshwork of desmin filaments in the cytoplasm and in close association with the plasma membrane. These observations suggest that intermediate filaments play a role in the maintenance of the morphology of electrocytes and, as an intracellular meshwork spanning the width of the cell, they may contribute to the organization of the intracellular compartments.
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  • 28
    ISSN: 1432-0878
    Keywords: Key words: Innervation ; Heart ; Intracardiac neurone ; Nitric oxide ; Immunocytochemistry ; Electron microscopy ; Rat (Sprague Dawley) ; Guinea-pig (Dunkin Hartley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity of intracardiac neurones in the rat and guinea-pig was studied at the ultrastructural level. While some nitric oxide synthase-containing intracardiac neurones were very heavily labelled, with electron-dense immunoprecipitate distributed throughout the neuronal cell bodies and their processes, most of the labelled neurones exhibited a lighter and more patchy distribution of nitric oxide synthase-immunoreactive material. Synapses made by nitric oxide synthase-negative nerve fibres with labelled intracardiac neurones were seen. Conversely, many nitric oxide synthase-containing nerve fibres that made synaptic contacts with unlabelled intracardiac neurones were also observed. Some small granule-containing cells were nitric oxide synthase-immunoreactive and were associated with unlabelled nerve terminals, while non-immunoreactive small granule-containing cells that were innervated by nitric oxide synthase-immunoreactive nerves were also seen. Small patches of osmiophilic electron-dense material were observed in the cytoplasm of NADPH-diaphorase-positive intracardiac neurones. This is the first description of the ultrastructural distribution of nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity in a subpopulation of intracardiac neurones of rat and guinea-pig heart and provides further evidence in support of a role for nitric oxide in the local control of the heart by intrinsic neurones.
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  • 29
    ISSN: 1432-0878
    Keywords: Key words: Ocellus ; Photoreceptor cells ; Retinal degeneration ; Subrhabdomeric cisternae ; Electron microscopy ; Immuno-electron microscopy ; Phosphatidylinositol transfer protein ; Drosophila melanogaster (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. We studied the structure of ocellar photoreceptor cells of Drosophila melanogaster, particularly the subrhabdomeric cisternae which our previous studies have shown to be essential structures for turnover of photoreceptive membranes in compound eyes. Each ocellus contained elongated photoreceptor cells with rhabdomeres positioned distally. In the subrhabdomeric regions, endocytotic invaginations were frequently observed, suggesting active turnover of photoreceptive membranes. In the vicinity of the photoreceptive microvilli, membranous structures similar to the subrhabdomeric cisternae in compound eyes were observed. These membranous structures were immunopositive for the rdgB protein, a phosphatidylinositol transfer protein that is localized to the subrhabdomeric cisternae in compound eyes. The ocellar photoreceptor cells of the retinal degeneration mutants (rdgA,B) were also studied. In these mutants, retinal degeneration has been reported to start, in compound eyes, with the disappearance of the subrhabdomeric cisternae. We found that the ocellar subrhabdomeric cisternae also disappear during the initial stage of retinal degeneration. From these observations, we conclude that the mechanism of photoreceptive membrane turnover in ocellar photoreceptor cells involves the rdgB and probably the rdgA proteins which are associated with subrhabdomeric cisternae, as is the case for photoreceptive membrane turnover in compound eyes.
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  • 30
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    Cell & tissue research 284 (1996), S. 125-142 
    ISSN: 1432-0878
    Keywords: Key words: Chordotonal organ ; Scolopidium ; Mechanoreceptor ; Sensilla ; Electron microscopy ; Phylogenesis ; Teleogryllus commodus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. More than 50 chordotonal sensilla, or scolopidia, embedded entirely in the integument were found in each side of the genital chamber wall in the female cricket, Teleogryllus commodus. Their cell bodies lie among the epidermal cells, and the tips of their dendrites terminate in the cuticle. About half of them contain two sensory cells (two-cell scolopidium), the others only one (one-cell scolopidium). The sensory cell in the one-cell scolopidium is the type-1 cell. In the two-cell scolopidium one is type-1 and the other type-2. Regardless of the number of sensory cells, they are all amphinematic. In the two-cell scolopidium only the type-2 dendrite, rich in microtubules, penetrates into the cuticle, bifurcates and terminates in the tube enclosed by an attachment cell; the type-1 never extends into the cuticle. On the other hand, the type-1 cell in the one-cell scolopidium projects its apex into the cuticle. The unique topography and structure of these scolopidia lead to the following hypothesis about the phylogenetic relationship between the scolopidia and other kinds of sensilla: the type-1 scolopidial sensory cell buried in the integument may be the original model, which through the loss of the long regular axoneme has given rise to type-2 cells. Modification of the apical region, the tubular body or ramification, may have lead to the cuticular sensilla corresponding to the development of the cuticular apparatus, and the scolopidia may have been withdrawn into the body cavity to form ordinary chordotonal organs.
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  • 31
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    Methods in cell science 18 (1996), S. 15-18 
    ISSN: 1573-0603
    Keywords: Angiogenesis ; Endothelial cells ; Angiogenesis analysis ; Electron microscopy ; Cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Determination of the structural characteristics of angiogenesis requires a procedure protective of the forming vascular fibers and the endothelial cell monolayer exhibiting cord formation. This report describes in situ fixation of angiogenic cultures in 96-well plates and the subsequent double embedding processing for electron microscopy. Cross sections of the monolayer are obtained without damage of the incipient capillaries.
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  • 32
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    European archives of oto-rhino-laryngology and head & neck 253 (1996), S. 205-213 
    ISSN: 1434-4726
    Keywords: Nasal neuropeptides ; Perennial rhinitis ; Mucosal glands ; Immunofluorescence ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to study morphological effects on the nasal and sinus mucosa, New Zealand White rabbits underwent either unselective, regional sectioning of sensory and parasympathetic nerve branches or topical treatment of the mucosa with capsaicin. Ten days after treatment, mucosal specimens were analyzed by light and electron microscopy. Immunohistochemistry was used to evaluate neuropeptides present, in particular substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and neuropeptide Y. In surgically denervated rabbits, mucosal glands were found to be enlarged and contained an increased number of zymogen granules having a bipartite substructure. Topical capsaicin application caused localized epithelial changes in the sinus mucosa and maxilloturbinal region of the nose, including clotting of cilia and an increased number of goblet cells. Reduced amounts of all neuropeptides investigated were found in the surgically denervated animals, while topical capsaicin treatment had only marginal effects on the mucosal neuropeptide content. The morphological changes observed after surgical denervation suggest an imbalance between neural stimulation and secretory capacity of the mucosal glands. These findings could explain the difference in clinical effect noted between sectioning of the vidian nerve and topical treatment with capsaicin in patients with perennial rhinitis.
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  • 33
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    Medical molecular morphology 29 (1996), S. 7-12 
    ISSN: 1860-1499
    Keywords: Keratin spherules ; Cholesterol crystals ; Epidermal cyst ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Numerous, small spherical inclusions with laminated horn-like material were observed in an epidermal cyst from the left earlobe of a 52-year-old Japanese man. They were examined by light and electron microscopy. Positive reactions obtained by staining with both oil red-O and anti-pankeratin antibody led to the conclusion that the inclusions consisted of lipid-rich keratin spherules. These were thought to have been formed in the hydrophobic regions of keratin proteins and non-polar lipids in the hydrophilic milieu of the cyst. Electron microscopy revealed cholesterol crystals within the spherules, suggesting that cholesterol had been slowly isolated from the lipoproteins and the membrane components of the cornified cells included in the lipid-rich keratin spherules, and had gradually become concentrated, finally appearing in the spherules as cholesterol crystals. With further study, a more detailed understanding of this process may throw some light on the development of gallstones and atheroembolism.
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  • 34
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    Medical molecular morphology 29 (1996), S. 37-42 
    ISSN: 1860-1499
    Keywords: Melkersson-Rosenthal syndrome ; Facial nerve sheath ; Electron microscopy ; Collagen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To our knowledge, this is the first report of electron microscopic findings in the facial nerve sheath in Melkersson-Rosenthal syndrome. We performed decompression of the right facial nerve on a patient with Melkersson-Rosenthal syndrome. A part from the facial nerve sheath was obtained and observed with a transmission electron microscope. There were some fibers 180 to 200 nm in diameter which had obscure cross bandings, and also accumulations of these degenerated fibers among normal collagen fibers.
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  • 35
    ISSN: 1860-1499
    Keywords: Placental site trophoblastic tumor ; Intermediate trophoblast ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A rare placental site trophoblastic tumor (PSTT) in a 39-year-old female was studied. This tumor, protruding into the uterine cavity, was histologically similar to tumors in previously reported cases of PSTT. Ultrastructurally, the characteristic finding was the presence of perinuclear filaments. Also, the tumor cells were strongly positive for hPL by immunohistochemical method. These findings suggest that this was a tumor caused by neoplastic proliferation of the extravillous intermediate trophoblast.
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  • 36
    ISSN: 1860-1499
    Keywords: Long-spacing collagen ; Intramuscular nerves ; Neuromuscular diseases ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We examined the incidence and morphological features of long-spacing collagen (LSC) in the intramuscular nerves of biopsied muscle tissues from patients with various neuromuscular diseases. LSC was found in 12 of 55 cases (21.8%). The muscles in all cases with LSC showed typical neurogenic changes at the light microscopic level. At the electron microscopic level, LSC was seen either in the vicinity of the basement membranes of the perineurium or Schwann cells, or running among the collagen fibrils. A close relationship between the form (compact or solitary type) and the periodicity of collagen was not confirmed.
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  • 37
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    Journal of Cellular Biochemistry 60 (1996), S. 4-11 
    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: Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals. © 1996 Wiley-Liss, Inc.
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  • 38
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    Journal of Cellular Biochemistry 60 (1996), S. 12-17 
    ISSN: 0730-2312
    Keywords: bcl-2 gene ; localization ; apoptosis ; antioxidants ; oxidative stress ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The bcl-2 gene has a unique function among mammalian oncogenes as a negative regulator of apoptosis. Its expression pattern in embryonic and adult tissues is consistent with a role in maintaining in vivo survival of specific cell types.The biochemical function of bcl-2 is unknown, but its localization to mitochondrial and microsomal membranes suggests several possibilities, bcl-2 is protective against oxidative stress in mammalian cells and can be replaced by antioxidants in a factor-deprivation model of apoptosis. These results are consistent with a model of apoptotic death involving oxidative stress in a central pathway.The recent discovery of several bcl-2-related genes, some of which also inhibit apoptosis and others that unexpectedly promote apoptosis, has shed new light on several aspects of bcl-2 action. © 1996 Wiley-Liss, Inc.
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  • 39
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    Journal of Cellular Biochemistry 60 (1996), S. 33-38 
    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.
    Additional Material: 3 Ill.
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  • 40
    ISSN: 0730-2312
    Keywords: BCL-2 gene ; Bcl-2 protein ; homologs ; homo- and heterotypic dimers ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases. © 1996 Wiley-Liss, Inc.
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  • 41
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    Journal of Cellular Biochemistry 60 (1996), S. 61-82 
    ISSN: 0730-2312
    Keywords: protein kinases ; cyclins ; nuclear import ; NLS ; acidic domains ; cell cycle ; phosphatases ; p34cdc2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Karyophilic and acidic clusters were found in most nonmembrane serine/threonine protein kinases whose primary structure was examined. These karyophilic clusters might mediate the anchoring of the kinase molecules to transporter proteins for their regulated nuclear import and might constitute the nuclear localization signals (NLS) of the kinase molecules. In contrast to protein transcription factors that are exclusively nuclear possessing strong karyophilic peptides composed of at least four arginines (R) and lysines (K) within an hexapeptide flanked by proline and glycine helix-breakers, protein kinases often contain one histidine and three K + R residues; this is proposed to specify a weak NLS structure resulting in the nuclear import of a fraction of the total cytoplasmic kinase molecules as well as in their weak retention in the different ionic strength nuclear environment. Putative NLS peptides in protein kinases may also contain hydrophobic or bulky aromatic amino acids proposed to further diminish their capacity to act as strong NLS. Most kinases lacking karyophilic clusters (c-Mos, v-Mos, sea star MAP, and yeast KIN28, SRA1, SRA3, TPK1, TPK2) also lack acidic clusters, which is in contrast to most kinases containing both acidic and karyophilic peptides; this and the presence of R/K clusters in the transporter proteins supports a role of acidic clusters on kinases in nuclear import. Cyclins B lack karyophilic signals and are proposed to be imported into nuclei via their association with Cdc2. © 1996 Wiley-Liss, Inc.
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  • 42
    ISSN: 0730-2312
    Keywords: protein kinase FA/GSK-3α ; PKC inhibition ; calphostin C ; down-regulation ; carcinoma dedifferentiation/progression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The signal transduction mechanism of protein kinase FA/GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa/GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1-10 μM calphostin C (IC50, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa/GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa/GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC50, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa/GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa/GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa/GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA/GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.
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  • 43
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    Journal of Cellular Biochemistry 60 (1996), S. 363-378 
    ISSN: 0730-2312
    Keywords: cyclin D1 function ; CDK activity ; pRB phosphorylation ; G1 phase ; cell cycle control ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent ∼ 3 h after the increase in cyclin D1 protein, and ∼ 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression. © 1996 Wiley-Liss, Inc.
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  • 44
    ISSN: 0730-2312
    Keywords: heregulin ; transformation ; erb B-2 ; c-Ha-ras ; mammary cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Heregulin β1 was found to stimulate the anchorage-dependent, serum-free growth of nontransformed human MCF-10A mammary epithelial cells. Unlike epidermal growth factor, transforming growth factor α, or amphiregulin, heregulin β1 was also able to induce the anchorage-independent growth of MCF-10A cells. In contrast, the anchorage-dependent, serum-free growth of c-Ha-ras or c-erb B-2 transformed MCF-10A cells was unaffected by heregulin β1, whereas heregulin β1 was able to stimulate the anchorage-independent growth of these cells. c-Ha-ras or c-erb B-2 (c-neu) transformed MCF-10A or mouse NOG-8 mammary epithelial cells express elevated levels of 2.5, 5.0, 6.5, 6.8, and 8.5 kb heregulin mRNA transcripts and/or synthesize cell-associated 25, 29, 50, and 115 kDa isoforms of heregulin. Since the MCF-10A cells and transformants also express c-erb B-3, these data suggest that endogenous heregulin might function as an autocrine growth factor for Ha-ras or erb B-2 transformed mammary epithelial cells. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
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  • 45
    ISSN: 0730-2312
    Keywords: ecto-enzyme ; ALP inhibitor ; Ca incorporation ; glycosylphosphatidylinositol-anchored proteins ; PI-PLC ; bone differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.
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  • 46
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 521-528 
    ISSN: 0730-2312
    Keywords: myosin heavy chains ; smooth muscle ; alternative splicing ; contractility ; myosin light chains ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca2+/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.
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  • 47
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 9-17 
    ISSN: 0730-2312
    Keywords: antiestrogen ; human breast cancer ; programmed cell death ; tamoxifen ; TGF-β1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-β1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-β was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-β. © 1996 Wiley-Liss, Inc.
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  • 48
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 39-47 
    ISSN: 0730-2312
    Keywords: α2M ; PAF ; RBF ; PKC ; lyso-PAF acetyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The binding of receptor-recognized forms of α2-macroglobulin (α2M) to macrophage α2M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca2+ mobilization. In this report, we demonstrate that ligation of the macrophage α2M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α2M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α2M+, stimulated macrophage synthesis of PAF from [3H]acetate, [3H]methylcholine, and 1-O-[3H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α2M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [3H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65-70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α2M-methylamine. By contrast, when [3H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α2M-methylamine. Both α2M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α2M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α2M to regulate levels of PAF suggests that α2M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α2M-proteinase complexes. © 1996 Wiley-Liss, Inc.
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  • 49
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 109-117 
    ISSN: 0730-2312
    Keywords: aggregin ; chemical modification ; ADP-induced platelet responses ; NBD-Cl ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 50
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 72-80 
    ISSN: 0730-2312
    Keywords: hypoxia ; S-adenosylmethionine ; DNA methylation ; hypomethylation ; t-RNA methyltransferase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Young rats were maintained in a 10% oxygen atmosphere for 2, 6, and 10 days and administered normal rat chow and water ad libitum. Thereafter, their hepatic S-adenosyl-L-methionine (AdoMet) and activity and mRNA levels of AdoMet synthetase were assayed. AdoMet levels decreased by 45% after 10 days; hepatic AdoMet synthetase also declined by ∼40%. In rats with low hepatic AdoMet, the mRNA level of AdoMet synthetase also declined by up to 80%. No significant change in AdoMet or AdoMet synthetase was noted in pair-fed normoxic rats. DNA hypomethylation was determined in terms of incorporation of [3H]methyl of AdoMet incorporated at unmethylated sites in DNA in reactions mediated by methylases Hpall and Sssl. As compared to the normal hepatic DNA, [3H]methyl group incorporation in the 10-day hypoxic DNA was almost double in the Hpall-mediated reaction and ∼10-fold in the Sssl-mediated reaction. Hepatic tRNA methyltransferase activity doubled after 10 days of hypoxia. However, hypoxic rats showed no detectable mRNA transcripts for c-myc and c-fos oncogenes on Northern blot analysis. These observations show that because of subnormal activity of AdoMet synthetase, hypoxic liver is depleted of AdoMet, even when the animals are administered a complete diet. However, unlike rats on chronic lipotrope-deficient diets, hypoxic rats on a complete diet show no aberrant expression of oncogenes. © 1996 Wiley-Liss, Inc.
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  • 51
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    Journal of Cellular Biochemistry 61 (1996), S. 31-38 
    ISSN: 0730-2312
    Keywords: cell density ; DNA synthesis ; Mr receptor substrates ; IRS-1 protein ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high Mr cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 105 to 3.2 × 106 per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high Mr receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high Mr proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.
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  • 52
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 48-60 
    ISSN: 0730-2312
    Keywords: nuclear pore structure ; digitonin permeabilization ; immunofluorescence ; coiled-coil proteins ; Tpr ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed. © 1996 Wiley-Liss, Inc.
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  • 53
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    Journal of Cellular Biochemistry 61 (1996), S. 127-138 
    ISSN: 0730-2312
    Keywords: β1 integrin ; β7 integrin ; α/β integrin subunit association ; VLA-4/VCAM adhesion ; integrin surface expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.
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  • 54
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    Journal of Cellular Biochemistry 61 (1996), S. 230-237 
    ISSN: 0730-2312
    Keywords: retinoic acid ; retinol ; binding ; transglutaminase ; hepatic ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: When rat liver epithelial cells were exposed to retinoic acid or retinol for 24 hr, the levels of transforming growth factor-β (TGF-β) receptors were reduced in a dose-dependent way. The decrease appeared after 12 hr of incubation with the retinoids and binding levels remained low until 24 hr after the removal of the molecules. Retinoid treatment induced a fourfold enhancement of transglutaminase (TGase) activity in the cell membranes, and cystamine, an inhibitor of TGase, prevented the decrease of the receptors. Neutralization of TGF-β by a monoclonal antibody did not suppress the decrease of the binding levels, indicating that decreased TGF-β binding capacity was not due merely to the internalization of ligand-bound receptors promoted by a stimulation of TGF-β synthesis. Thus, retinoid treatment resulted in an intense disappearance of the functional receptors from the membranes that seemed to be mediated by increased TGase activity. This phenomenon can represent a strong signal attenuation for TGF-β following retinoid exposure. © 1996 Wiley-Liss, Inc.
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  • 55
    ISSN: 0730-2312
    Keywords: human hepatoma ; dedifferentiation/progression ; PDPK ; overexpression ; kinase FA/GSK-3α ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P 〈 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 ± 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 ± 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 ± 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3α is due to overexpression of the protein. Elevated kinase FA/GSK-3α expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be ∼fivefold overexpressed in well differentiated hepatoma and ∼13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3α is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3α is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression. © 1996 Wiley-Liss, Inc.
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  • 56
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    Journal of Cellular Biochemistry 61 (1996), S. 246-254 
    ISSN: 0730-2312
    Keywords: marrow stromal cells ; cell morphogenesis ; attachment ; ECM ; mRNA expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.
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  • 57
    ISSN: 0730-2312
    Keywords: adhesion ; migration ; protease ; lymphocyte ; immunity ; connective tissue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human T lymphoblastoma cells of the CD4+ 8+ Tsup-1 line, that express alpha4 and alpha5 but not alpha6 integrins of the beta1 family, and CD4+ human blood T cells bind vasoactive intestinal peptide (VIP) with high affinity, leading to increased adherence, secretion of matrix metalloproteinases (MMPs), and chemotaxis. VIP-enhanced adherence of T cells to fibronectin was inhibited significantly by neutralizing monoclonal antibodies to beta1 〉 alpha4 〉〉 alpha5, but not to alpha6. Antibodies to beta1 and alpha4 suppressed to a similarly significant extent VIP stimulation of both MMP-dependent T cell chemotaxis through fibronectin-enriched Matrigel and T cell degradation of 3H-type IV collagen in the Matrigel, without affecting VIP-evoked secretion of MMP by suspensions of T cells. The lesser inhibition of VIP-enhanced adherence of T cells to fibronectin by anti-alpha5 antibody, than antibodies to beta1 or alpha4 chains, was associated with lesser or no suppression of MMP-dependent T cell chemotaxis through Matrigel and T cell degradation of type IV collagen in the Matrigel in response to VIP. Specific beta1 integrins thus mediate interactions of stimulated T cells with basement membranes, including adherence, localized digestion by MMPs, and chemotactic passage, that promote entry of T cells into extravascular tissues. © 1996 Wiley-Liss, Inc.
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  • 58
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    Journal of Cellular Biochemistry 61 (1996), S. 459-466 
    ISSN: 0730-2312
    Keywords: adenylyl cyclase ; BAT3 ; cytoskeleton ; RAS ; signaling ; yeast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously identified human CAP, a homolog of the yeast adenylyl cyclase - associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts. © 1996 Wiley-Liss, Inc.
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  • 59
    ISSN: 0730-2312
    Keywords: basic helix-loop-helix ; interleukin-1 ; interleukin-3 ; granulocyte-macrophage colony-stimulating factor ; progenitor ; transcription factor ; c-kit ligand ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The basic helix-loop-helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl-1, E12/E47, and Id-1, in individual lineage-defined progenitors and hematopoietic growth factor - dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage-defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT-PCR. SCL and E12/E47 were expressed in the vast majority of tri-, bi-, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl-1 showed a more restricted pattern of expression, although expression was seen in some bi- and unilineage progenitors. No expression was detected in erythroid, erythroid-megakaryocyte-macrophage, macrophage-neutrophil, macrophage, or megakaryocytic progenitors. Id-1, an inhibitory bHLH transcription factor, was also widely expressed in all bi- and unilineage progenitors; only the trilineage erythroid-megakaryocyte-macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id-1, but not E12/E47, mRNA was increased in FDC-P1 and MO7E hematopoietic cell lines after exposure to IL-3 or GM-CSF, Id-1, E12, and lyl-1 showed marked variation at different points in cell cycle in isoleucine-synchronized FDC-P1 cells. These results suggest that SCL, lyl-1, E12/E47, and Id-1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle. © 1996 Wiley-Liss, Inc.
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  • 60
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    Journal of Cellular Biochemistry 61 (1996), S. 493-501 
    ISSN: 0730-2312
    Keywords: basement membrane ; cell binding ; epidermolysis bullosa ; extracellular matrix ; gene knock-out ; integrin ; laminin ; muscular dystrophy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been found in a number of other proteins, and data on these proteins may be informative in terms of structure-function relationships in laminin. Finally, a large body of data has accumulated on the structure and activities of proteolytic fragments, recombinant fragments, and synthetic peptides from laminin. The proposed activities of these domains can now be confirmed and extended by in vivo experiments. © 1996 Wiley-Liss, Inc.
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  • 61
    ISSN: 0730-2312
    Keywords: bone resorption ; tyrphostins ; genistein ; herbimycin ; osteoporosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P 〈 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.
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  • 62
    ISSN: 0730-2312
    Keywords: CoA-independent transacylase ; phospholipase D ; subcellular localization ; neutrophils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.
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  • 63
    ISSN: 0730-2312
    Keywords: FGF ; receptors ; internalization ; photoactivable cross-linker ; heparan sulfate proteoglycans ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.
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  • 64
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 275-289 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; HeLa S3 cells ; 2-D gel electrophoresis ; heterogeneous nuclear ribonucleoproteins ; B23 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.
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  • 65
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 314-324 
    ISSN: 0730-2312
    Keywords: M1 cell ; heme oxygenase ; transcription ; H2O2 ; TPA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: It has long been known that heme oxygenase (HO) is a key enzyme in heme catabolism and recently it was also found to acts as an oxidative stress protein to produce carbon monoxide (CO), which has similar actions to those of nitrogen monoxide (NO). Therefore, we examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during their differentiation into macrophages. Since the promoter region of this gene is known to have a TPA-responsive element (TRE), its expression might be regulated by a C-kinase signal transduction pathway. Then we investigated the activation of the HO gene after treatment of M1 cells with TPA and inhibitors of C-kinase. When M1 cells were treated with TPA, they differentiated into macrophage-like cells. Upon treatment with TPA, H2O2 was produced first, the nuclear proto-oncogenes fos and jun were activated, and then the HO gene was activated. The extent of transcriptional activation of the fos, jun, and HO genes in M1 cells treated with TPA was reduced by a specific inhibitor of C-kinase and a scavenger of oxygen radicals. When M1 cells were treated with H2O2 essentially the same level of transcription of the HO gene was observed, but the extent of transcriptional activation of the fos and jun genes was about half of the treatment with TPA. Super-shift assays using the TRE of the HO gene revealed that the Fos and Jun proteins from nuclei of M1 cells treated with TPA bound to the TRE, and same assays using DNA with the NF-kB motif also revealed that the active NF-kB protein from M1 cells treated with H2O2 or TPA also bound to the corresponding motif. These results strongly suggest that the HO gene in M1 cells is activated by TPA through a production of H2O2, an oxidative activation pathway of NF-kB, and a signal-transduction pathway that involves C-kinase during the differentiation of macrophages that occurs upon treatment with TPA. © 1996 Wiley-Liss, Inc.
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  • 66
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 172-180 
    ISSN: 0730-2312
    Keywords: chromatin structure ; nuclear matrix ; transcriptional activation ; replication ; recombination ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrety of the genome. This function of wild-type p53 as “guardian of the genome” is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function. © 1996 Wiley-Liss, Inc.
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  • 67
    ISSN: 0730-2312
    Keywords: dexamethasone ; actin ; polymerization ; Ishikawa cells ; cAMP ; actinomycin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total-actin ratio in a time- and dose-dependent manner. Specifically, in untreated Ishikawa cells the G/total-actin ratio was 0.48 ± 0.01 (n = 26). It became 0.35 ± 0.01 (n = 13, P 〈 0.01) following exposure to 10-7 M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G-actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F-actin content by 44% (n = 6, P 〈 0.001) in cells treated with dexamethasone (10-7 M, 15 min). The rapid dexamethasone-induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10-7 M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non-transcriptional pathways, since actinomycin D did not block the dexamethasone-induced actin polymerization. In addition, cell treatment with 10-7 M dexamethasone for 15 min fully reversed the forskolin-, but not the 8-bromo-cAMP-induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10-7 M dexamethasone (n = 4, P 〈 0.01). In conclusion, our results showed that dexamethasone induces rapid, time-, and dose-dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids. © 1996 Wiley-Liss, Inc.
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  • 68
    ISSN: 0730-2312
    Keywords: monocyte chemoattractant protein-1 ; gene expression ; pig artery ; balloon injury ; monocyte/macrophages ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are potent chemokines which attract circulating monocytes and neutrophils respectively to inflamed tissues. JE/MCP-1 gene expression has been previously studied in rabbit aortae after endothelial denudation and the rapid appearance of this transcript was thought to precede emigration of phagocytes. We now report MCP-1 gene expression following de-endothelialization of iliac arteries in the pig, a species which can develop spontaneous atherosclerosis. Using Northern blot analysis, we demonstrated that MCP-1 mRNA was rapidly induced in pig arteries at 2 h and continued to increase to reach a maximum at 8 h before returning to low levels at 16-24 h after injury. The increase seen for MCP-1 mRNA at 8 h was also observed for IL-8 mRNA but was not apparent for growth-related gene expressions, urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor-1 (PAI-1). Since smooth muscle cells, endothelial cells, and phagocytes are all capable of expressing MCP-1, we examined pig arteries for immunostaining using a monoclonal antibody to human MCP-1 (5D3-F7). At 8 h after injury, the predominant cell type staining positive for MCP-1 was the monocyte/macrophage. Staining was also observed in occasional scattered neutrophils, but MCP-1 protein could not be detected in smooth muscle cells or on extracellular matrix within the sensitivity constraints posed by our methodology. Our results are consistent with invading monocyte/macrophages having a major input into the production of this chemokine in the arterial wall following injury. The fact that MCP-1 expression accompanied monocyte/macrophage presence in damaged artery, rather than preceding it, is suggestive that continued MCP-1 expression is required for functions other than chemoattraction. © 1996 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 69
    ISSN: 0730-2312
    Keywords: GLRP ; T-lymphocyte ; immune response ; central nervous system ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-β2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-β2 transcription correlates with the loss of binding activity for an 80 kDa glial labile repressor protein, GLRP, to a responsive region within the TGF-β2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-β2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-β2 to regulate the immune response. © 1996 Wiley-Liss, Inc.
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  • 70
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 454-466 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; histone H5 ; transcription ; transcription factors ; erythroid development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression. © 1996 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 71
    ISSN: 0730-2312
    Keywords: adhesion ; breast cancer cells ; thrombospondin ; receptors ; proteoglycans ; heparin-binding peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 μg/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 μg/ml) had little or no effect. Herapin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two β-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity. © 1996 Wiley-Liss, Inc.
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  • 72
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 506-515 
    ISSN: 0730-2312
    Keywords: heat shock ; pre-rRNA processing ; S-100 extract ; U3 snoRNA ; 3′ processing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effect of heat shock on pre-rRNA processing at the primary site within external transcribed spacer region 1 (ETS1) was studied in S-100 extract derived from mouse lymphosarcoma cells. In vivo labeling with [32P]orthophosphate showed that the synthesis of the rRNA precursor and its processing to 28S and 18S rRNAs were inhibited significantly due to heat shock. The processing activity was reduced by 50% at 1 h and was completely blocked following 2-h exposure of cells at 42°C. Mixing S-100 extracts from the control and heat-treated cells did not affect the processing activity in the control extract, which proves the absence of a nuclease or other inhibitor(s) of processing in the extract from the heat-shocked cells. Heat shock did not affect interaction between pre-rRNA and U3 snoRNA, a prerequisite for the processing at the primary site, but significantly altered RNA-protein interaction. Three polypeptides of 200, 110, 52 kDa that specifically cross-link to pre-rRNA spanning the primary processing site were inactivated after heat shock. Hyperthermia did not alter 3′ end processing of SV40L pre-mRNA. © 1996 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 73
    ISSN: 0730-2312
    Keywords: osteosarcoma ; chondrosarcoma ; GCT ; oncogene alterations ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors - osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients) - in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease. © 1996 Wiley-Liss, Inc.
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  • 74
    ISSN: 0730-2312
    Keywords: Src kinase ; mercuric chloride ; redox ; sulfhydryl group ; receptor polymerization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl2) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg2+ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl2 at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl2-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.
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  • 75
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 162-173 
    ISSN: 0730-2312
    Keywords: Topo IIα ; Topo IIβ ; interphase ; mitosis ; mitogenic stimulation ; nucleoplasm ; nucleolus ; lymphocytes ; HeLa ; immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have carried out immunofluorescence labelling of two human cell types, HeLa cells and peripheral blood lymphocytes, prepared by several different fixation/permeabilization protocols using a variety of antibodies against DNA Topoisomerase II (Topo II). We have found that the distribution of Topo IIα was overall similar during interphase and mitosis to that previously reported, regardless of antibody and of sample preparation. On the other hand, the interphase distribution of Topo IIβ was quite variable, depending both on the antibody and on the method used to prepare the sample. Our interpretation of the data is that, like Topo IIα, Topo IIβ is primarily a nucleoplasmic protein, but that unlike Topo IIα, small amounts are also associated with intranucleolar chromatin. © 1996 Wiley-Liss, Inc.
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  • 76
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 185-198 
    ISSN: 0730-2312
    Keywords: extracellular matrix ; remodeling ; collagenase ; collagen ; dilated cardiomyopathy ; congestive heart disease ; end-stage heart failure ; matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; differential display mRNA analysis ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13-21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP-1). Northern blot analysis using MMP-1 cDNA probe indicated that MMP-1 was induced three- to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP-1 and MMP-1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P 〈 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P 〈 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP-1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP-1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic-fibroblast cell ECM-proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy. © 1996 Wiley-Liss, Inc.
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  • 77
    ISSN: 0730-2312
    Keywords: regulation of transcription ; control of proliferation ; vitamin D3 analogues ; vitamin D3 receptor ; limited protease digestion assay ; lymphocytes ; breast cancer cells ; promoter selectivity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues. © 1996 Wiley-Liss, Inc.
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  • 78
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    Journal of Cellular Biochemistry 63 (1996), S. 239-251 
    ISSN: 0730-2312
    Keywords: lymphocyte activation ; Krebs cycle ; energy metabolism ; immunosuppressives ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.
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  • 79
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    Journal of Cellular Biochemistry 63 (1996), S. 268-279 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; mitosis ; Drosophila embryo ; monoclonal antibody ; spindle formation ; nucleus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.
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  • 80
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    Journal of Cellular Biochemistry 63 (1996), S. 311-319 
    ISSN: 0730-2312
    Keywords: protein phosphatase 2A ; endothelial cells ; cyclic strain ; proliferation ; okadaic acid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.
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  • 81
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    Journal of Cellular Biochemistry 63 (1996), S. 366-373 
    ISSN: 0730-2312
    Keywords: capsule ; lipid droplet ; Leydig cell ; monoclonal antibody ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.
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  • 82
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    Journal of Cellular Biochemistry 63 (1996), S. 453-462 
    ISSN: 0730-2312
    Keywords: FBPase ; gluconeogenesis ; perinuclear association ; metabolic zonation ; immunolocalization ; subcellular fractionation ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-P2-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense. © 1996 Wiley-Liss, Inc.
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  • 83
    ISSN: 0730-2312
    Keywords: transcription initiation ; CpG island ; transcription factor AP2 ; transcription factor Sp1 ; osteoblasts ; differentiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transforming growth factor (TGF-β) binds several discrete membrane proteins. Of these, a type I receptor appears indispensable for signal transduction. Previous examination of TGF-β receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-β function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-β type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-β type I receptor in bone. © 1996 Wiley-Liss, Inc.
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  • 84
    ISSN: 0730-2312
    Keywords: protein kinase C ; Drosophila melanogaster ; embryonic neurons ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Embryonic neurons were cultured from transgenic Drosophila melanogaster expressing a highly specific pseudosubstrate inhibitor of protein kinase C (PKC). Flies homozygous for this transgene, which is under the control of the yeast UAS promoter, were crossed to flies homozygous for the yeast heat shock inducible transcription factor GAL 4. Following heat shock, the progeny express the pseudosubstrate inhibitor at high levels. This strategy, which has the advantage of avoiding the non-specific effects of drugs, was used to study the role of PKC in process growth of cultured, differentiating neuroblasts. An external gold particle labeling procedure using a cell surface antigen expressed by mature neurons and processes was used to visualize neuronal processes directly in the scanning electron microscope. We observed that cell cultures expressing a low concentration of the pseudosubstrate inhibitor showed a significant decrease in the number of type I and II processes as compared to control cultures, while the proportions of neuroblasts, ganglion mother cells (GMCs), and mature neurons in the clusters were little affected. © 1996 Wiley-Liss, Inc.
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  • 85
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    Journal of Cellular Biochemistry 61 (1996), S. 18-25 
    ISSN: 0730-2312
    Keywords: osteoblasts ; calvaria ; invasion ; prostate ; PC-3 cells ; differentiation ; metastasis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells. © 1996 Wiley-Liss, Inc.
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  • 86
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    Journal of Cellular Biochemistry 61 (1996), S. 97-108 
    ISSN: 0730-2312
    Keywords: aggregin ; chemical modification ; ADP-induced platelet responses ; NBD-Cl ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I2). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [14C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [14C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.
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  • 87
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    Journal of Cellular Biochemistry 63 (1996), S. 108-111 
    ISSN: 0730-2312
    Keywords: duct carcinoma in situ ; nuclear grade necrosis ; prognostic features ; local recurrence ; invasive transformation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the last 6 years a number of non-randomized, predominantly single institutional trials of breast conservation therapy (BCT) with DCIS, have demonstrated that it constitutes a very heterogeneous group of diseases with markedly different risks of local recurrence and invasive transformation. There has been a consensus that DCIS, which exhibits a “comedo” morphology, generally defines a high risk group. Most studies, moreover, have identified the same two features, nuclear grade and necrosis, as contributing most significantly to prognosis [4-6]. Nuclear grade and necrosis have been identified as independent prognostic variables in several studies [5,6]. High nuclear grade DCIS which exhibits comedo necrosis defines the majority of all DCIS which will result in local recurrence and invasive transformation after BCT.Studies utilizing image cytometry, to determine ploidy and S-phase fraction and immunohistochemical studies of proliferation and oncogene distribution have shown a significant association with morphologically identified high nuclear grade and aneuploidy, high S-phase fraction or proliferation rate, presence of HER-2/neu and P53 oncogenes and absence of estrogen receptors. Generally the inverse of this association is seen with low nuclear grade DCIS. However, initial hopes that these adjunctive studies would identify subsets within the high nuclear grade group which might be more likely to recur have not been fulfilled. J. Cell. Biochem. 25S:108-111. © 1997 Wiley-Liss, Inc.
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  • 88
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    Journal of Cellular Biochemistry 63 (1996), S. 123-130 
    ISSN: 0730-2312
    Keywords: carcinogenesis ; predisposing mutation ; malignancy ; DNA testing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Identification of cohorts at genetic risk for cancer offers unique research opportunities to explore the steps in carcinogenesis, from the inheritance of a predisposing mutation to the development of preinvasive lesions or overt malignancy, and to evaluate interventions to modulate the carcinogenic process. However, cancer prevention strategies for most inherited cancer predisposition syndromes are of unproven benefit, and the potential for adverse psychosocial effects and employment or insurance discrimination associated with genetic testing is substantial. Thus testing for genetic cancer risk remains highly controversial, and the National Center for Human Genome Research and the American Society of Human Genetics advise DNA testing for presymptomatic identification of cancer risk only in the setting of a carefully monitored research environment.The commercial availability of predictive genetic testing, particularly for inherited susceptibility to cancer, has focused attention not only on the urgent need for research in cancer prevention for cohorts at genetic cancer risk but also on ethical considerations surrounding clinical prevention research in genetic risk groups. This paper addresses the interrelationship of ethical and scientific issues in conducting chemoprevention research in these cohorts, especially for those studies which require presymptomatic testing for specific gene mutations as a study entry criterion or as a criterion for stratification. Practical approaches to study design and implementation issues for chemoprevention research in genetic risk cohorts are discussed, emphasizing the interactions of ethical and scientific considerations at all levels of the research process. J. Cell. Biochem. 25S:123-130. © 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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  • 89
    ISSN: 0730-2312
    Keywords: acquired risk ; chemoprevention ; colon ; genetic risk ; neoplasia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The progressively abnormal development of epithelial cells prior to tumor development leads to widely differing chemopreventive approaches. The diversity of these approaches has resulted in different assays to measure the activities of the agents. To apply these assays to preclinical studies, we have developed rodent models in which different stages of evolution of colonic neoplasia are expressed. In one model mice carrying a truncated Apc allele with a nonsense mutation in exon 15 have been generated by gene targeting and embryonic stem cell technology (Apc1638 mice). These mice develop multiple gastrointestinal lesions including adenomas and carcinomas, focal areas of high grade dysplasia (FAD) and polypoid hyperplasias with FADS.The incidence of inherited colonic neoplasms has now been modulated by a chemopreventive regimen. Colonic lesions significantly increased in Apc1638 mice on a Western-style diet, compared to Apc1638 mice on AIN-76A diet which has lower fat content and higher calcium and vitamin D. These studies have also been carried out in normal mice, and have demonstrated without any chemical carcinogen that a Western-style diet induced colonic tumorigenesis. Modulation of cell proliferation has also been induced by Western-style diets in other organs including mammary gland, pancreas and prostate. These findings are leading to the development of new preclinical models for evaluating the efficacy of many classes of chemopreventive agents. J. Cell. Biochem. 25S:136-141. © 1997 Wiley-Liss, Inc.
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  • 90
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    Journal of Cellular Biochemistry 63 (1996), S. 156-164 
    ISSN: 0730-2312
    Keywords: carcinogenesis ; chemoprevention ; prostate cancer ; prostatic intraepithelial neoplasia ; prostatic neoplasms ; surrogate endpoint biomarkers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The most efficient strategy for chemoprevention clinical trials are short-term studies which focus on surrogate endpoint biomarkers (SEBs) in high-risk target populations. High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate cancer, and is found in a significant number of routine contemporary needle biopsies without cancer. The frequency and extent of PIN are decreased with androgen deprivation therapy, suggesting that it is a suitable endpoint biomarker for modulation. Potential SEBs for screening chemopreventive agents for prostate cancer in short-term Phase II trials include (1) histologic premalignant lesions, such as high-grade PIN; (2) biochemical markers, including prostate-specific antigen (PSA) serum concentration; and (3) morphometric markers, including nuclear texture, shape, and roundness; size and number of nucleoli; and number of apoptotic bodies; (4) proliferation markers, including MIB-1 and PCNA; (5) genetic markers, including nuclear DNA content (ploidy), oncogene c-erbB-2 (HER-2/neu) expression, fluorescence in situ hybridization for chromosome 8; and PSA-producing cells in the blood detected by reverse transcriptase polymerase chain reaction; and (6) differentiation markers, such as microvessel density as a determinant of angiogenesis. Each of these endpoint biomarkers is measured easily and accurately in serum or in tissue specimens such as formalin-fixed, paraffin-embedded needle biopsies, and may be modifiable by intervention. The clinical utility of these biomarkers as modulatable endpoints in prostate cancer chemoprevention needs to be demonstrated in future clinical trials. J. Cell. Biochem. 25S:156-164. © 1997 Wiley-Liss, Inc.
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  • 91
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    Journal of Cellular Biochemistry 62 (1996), S. 102-112 
    ISSN: 0730-2312
    Keywords: NDF ; estrogen receptor ; breast cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-β2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BP-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-β-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2-6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells. © 1996 Wiley-Liss, Inc.
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  • 92
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    Journal of Cellular Biochemistry 60 (1996), S. 508-520 
    ISSN: 0730-2312
    Keywords: chondrocyte ; porcine ; countercurrent centrifugal elutriation ; cartilage ; alkaline phosphatase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Countercurrent centrifugal elutriation was used to separate growth plate chondrocytes from porcine basing on their differences in sizes and densities. Eighteen fractions of cells with different sizes and densities were obtained. The mean cellular volumes increased progressively in each of successive fractions, and that increase was associated with specific phenotypic changes, such as biochemical differences in DNA synthesis, proteoglycan synthesis, and activities of alkaline phosphatase. Three distinct chondrocyte subpopulations with their unique characteristics were identified among the elutriated fractions. The resting chondrocytes were found to be small in size and quiescent. The hypertrophic chondrocytes were found to be large in size and metabolically active both in alkaline phosphatase and in proteoglycan productions. The proliferative chondrocytes exhibited a high DNA synthesis rate, and their sizes were found to be between those of the resting and hypertrophic chondrocytes. © 1996 Wiley-Liss, Inc.
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  • 93
    ISSN: 0730-2312
    Keywords: glycoprotein ; cell adhesion ; COLO 205 cell line ; affinity chromatography ; MUC1 mucin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A secreted MUC1 mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration. The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1β (IL-1β)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectin-expressing COS-1 cells and IL-1β-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing mucins having a MUC1 apoprotein, as shown by its size, and reactivity with the two anti-MUC1 apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUC1 mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUC1 mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUC1 mucin from cells. It is hypothesized that sialyl-Lewis a- and/or x-containing mucins, especially MUC1, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion. © 1996 Wiley-Liss, Inc.
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  • 94
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    Journal of Cellular Biochemistry 62 (1996), S. 165-171 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; phosphorylation ; protein kinase CK2 ; chromatin ; nuclear translocation ; prostate ; cell growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nuclear matrix (NM) is not only the structural basis for nuclear shape but also is intimately involved in nuclear functional activities. Among the modulatory factors that may affect these diverse activities are the signals that may influence the state or composition of the NM proteins. One such mechanism for altering the functional activity of at least some NM proteins may be the extent of their phosphorylation. Protein kinase CK2 appears to associate with NM and to phosphorylate a number of NM-associated proteins. Chromatin- and NM-associated CK2 is rapidly modulated by mitogenic signals. We propose that NM serves as a physiological anchor for nuclear signalling of protein kinase CK2 which may influence functions of NM such as transcription of active genes and growth. © 1996 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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  • 95
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    Journal of Cellular Biochemistry 62 (1996), S. 158-164 
    ISSN: 0730-2312
    Keywords: nuclear matrix ; mitosis ; mitotic apparatus ; matrix-associated proteins ; genome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert “passengers” or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle. © 1996 Wiley-Liss, Inc.
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  • 96
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 181-190 
    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.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 97
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 61-71 
    ISSN: 0730-2312
    Keywords: α2M* ; cAMP synthesis ; IP3 synthesis ; α1I3 ; conformational changes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Binding of receptor-recognized forms of tetrameric human α2-macroglobulin (α2M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP3) synthesis, and a concomitant rise in cytosolic free calcium ([Ca2+]i). The α2M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of α2M* also occurs to the low density lipoprotein receptor-related protein/α2M receptor (LRP/α2MR), but this binding does not induce signal transduction. Rat α1-inhibitor-3 (α1I3) is a monomeric member of the α-macroglobulin/complement superfamily. Like α2M, it can react with proteinases or methylamine which induces a conformational change causing activated α1I3 to bind to LRP/α2MR. We now report that α1I3-methylamine binds to the macrophage α2M* signaling receptor inducing a rapid rise in the synthesis of IP3 with a subsequent 1.5- to 3-fold rise in [Ca2+]i. α1I3-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native α1I3, like α2M, was unable to induce signal transduction. α1I3 forms a complex with α1-microglobulin, which has a distinct conformation from α1I3 and is recognized by LRP/α2MR. This complex also induces an increase in [Ca2+]i comparable to the effect of α1I3-methylamine on macrophages. It is concluded that activation of α1I3 by methylamine or binding of α1-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the α2M* signaling receptor, as well as for LRP/α2MR. © 1996 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 98
    ISSN: 0730-2312
    Keywords: mRNA sorting ; mRNA targeting ; urea cycle ; enzyme organization ; cell organization ; electron microscopy ; digoxigenin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/10 nm gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver “enrichment ratio” for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAS. © 1996 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 99
    ISSN: 0730-2312
    Keywords: Oxidative stress ; redox-state ; antioxidants ; extracellular matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; fibroblast ; polyoma virus transformation ; tumor ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Latent matrix metalloproteinases (MMPs) in normal myocardium are activated in end-stage heart failure. In vitro oxidized glutathione (GSSG) activates myocardial MMPs which contains a cysteine residue. In vivo GSSG induce the collagen lysis and cardiac dilatation. To assess whether thiol and non-thiol reducing agents have direct effect on the interstitial human heart fibroblast (HHF) proliferation and MMP expression, HHF and polyoma virus transformed fibroblast cells were cultured with or without the thiol-containing reduced (GSH) or oxidized (GSSG) glutathiones, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), and non-thiol ascorbic acid. After 100 μg/ml (∼0.3 mM) GSH or PDTC treatment the proliferative (synthetic) phenotype of transformed fibroblast cells was changed to quiescent (contractile) phenotype. Also, after GSH, PDTC, and ascorbic acid treatment the medium was then analyzed for MMP activity by zymography. The results indicate reduction in MMP expression in transformed fibroblast cells after GSH and PDTC treatments and no effect after ascorbic acid treatment. Based on reverse zymography, we observed the level of tissue inhibitor of metalloproteinase (TIMP) at a decreased level in transformed cells. The effect of the reducing agent at the gene transcription was measured by estimating mRNA (Northern blot analysis) of MMP and of TIMP in the cells that were cultured in medium in the presence and absence of GSH. These results indicate that GSH induces MMP-2 and MMP-1 expression in normal HHF and that GSH reduces MMP-2 and MMP-1 in transformed fibroblast cells. After the treatment, the TIMP-2 level was repressed in normal HHF and TIMP-2 level increased in transformed fibroblast cells. These events are dependent on the nuclear transcription factor activity on the collagenase promoter in normal HHF cells. On the other hand, in polyoma transform fibroblast cells these events are not dependent on this collagenase promoter. These results suggest that oxidative environment induces normal HHF cell proliferation, and the reducing agent decreases normal HHF cell proliferation by inducing MMP and repressing TIMP gene transcription. In transformed cells reducing agents inhibit MMP expression and increase TIMP levels, which suggests a role of antioxidants in preventing tumorigenesis. © 1996 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 100
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 182-193 
    ISSN: 0730-2312
    Keywords: glucocorticoid ; alkaline phosphatase ; osteopontin ; osteocalcin ; bone sialoprotein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10-10 M with maximum values achieved at 10-8 M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)2D3 during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. © 1996 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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