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  • 1995-1999  (6,973)
  • 1970-1974  (1,443)
  • Biochemistry and Biotechnology  (5,793)
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  • Ultrastructure
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  • 1
    ISSN: 1437-773X
    Keywords: Key words Heart ; Ultrastructure ; Capillaries ; Endothelium ; Stereology ; Cardioplegic solutions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In open heart surgery and transplantation, sufficient structural preservation of the myocardium immediately following cardioplegic arrest is a precondition for overcoming ischemia and for resumption of postischemic function. Therefore, we compared the protective effect of three clinically applied cardioplegic solutions with fibrillating and beating hearts using structural criteria. Left ventricular samples were taken from (1) beating, or (2) fibrillating or arrested hearts following coronary perfu-sion with (3) St. Thomas' Hospital solution, (4) histidine tryptophane ketoglutalate (HTK) (Custodiol), or (5) University of Wisconsin (UW) solution and fixed by immersion. Ultrastructural differences in the swelling of capillary endothelial cells and myocytes were quantitatively evaluated using stereological methods. Endothelial cells were somewhat more swollen after St. Thomas perfusion than those in beating and fibrillating hearts. HTK-arrested hearts showed significantly lower values for cellular edema than beating hearts. UW perfusion resulted in the (significantly) lowest degree of endothelial cell edema. Edematous changes in myocytes were significantly greater in St. Thomas-arrested hearts than in UW- or HTK-arrested hearts. Cardiomyocyte edema in beating and fibrillating hearts was comparable to that in St. Thomas-perfused hearts. Thus, the stereol-ogical analysis revealed significant differences between cardioplegic solutions in structural preservation of myocardial ultrastructure.
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  • 2
    ISSN: 1437-773X
    Keywords: Key words: Secretory carcinoma ; Breast ; Intracytoplasmic lumina ; Immnohistochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report a rare case of secretory carcinoma of the breast in a 50-year-old Japanese woman. The patient had been aware of a right breast tumor for 8 years, but had left it untreated. The tumor enlarged in size and became painful, and she visited our hospital. Breast carcinoma was diagnosed, and mastectomy was performed. Histopathological examination revealed features of a secretory carcinoma characterized by prominent secretory activity in the glandular and microcystic spaces, with some areas showing a follicular pattern resembling the thyroid gland. The secretory material was PAS-positive and immunohistochemically α-lactalbumin-positive. Ultrastructurally, the tumor cell contained many secretory vacuoles in the cytoplasm. In addition, extracellular and intracytoplasmic lumina were conspicuous; these were lined by microvilli projection and contained secretory material. By flow cytometric analysis, the DNA index was 1.14, which was diploid, showing relatively low proliferative activity.
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  • 3
    ISSN: 1437-773X
    Keywords: Key words KSHV ; HHV-8 ; TPA ; Ultrastructure ; Primary effusion lymphoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The ultrastructure of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) has not yet been fully elucidated, although some findings have been reported using primary effusion lymphoma (PEL) cell lines, KS-1, harboring no Epstein–Barr virus (EBV) coinfection. In the present study, detailed fine structural examination of KSHV/HHV-8 was performed after stimulation of the PEL-derived cell line KS-1 with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in vitro. While unstimulated KS-1 cells contained a small number of intranuclear virus particles associated with no extracellular mature particles, KS-1 cells stimulated with TPA produced many extracellular mature particles as well as intranuclear particles, in addition to interesting tubulo-reticular structures and aggregated tubular structures in vesicles. The induced intranuclear particles were empty, doughnut shaped, and dense cored, with outer and inner diameters of 100–110 nm and 60–70 nm, respectively. Dense-cored extracellular mature particles were 150–160 nm in diameter, and some contained doughnut-shaped cores, together with a few megaloviruses, 260 nm in outer diameter. These findings indicate that KS-1 cells treated with TPA can produce extracellular mature particles as well as intranuclear particles, which were proven to be KSHV/HHV-8.
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  • 4
    ISSN: 1437-773X
    Keywords: Key words pHGF ; HGF ; Acute liver injury ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The protective effect of hepatocyte growth-promoting factor (pHGF) against CCl4-induced acute hepatitis in rats was examined by light and electron microscopy. Hepatocyte growth-promoting factor, purified from infant pig liver in an active form, has been used clinically in patients with hepatitis in China. Four hours after administration of CCl4, a single dose of pHGF was administered intraperitoneally. Six hours after administration of CCl4, inhibition of CCl4-induced hepatic necrosis and hepatocytes with severely dilated endoplasmic reticula were evident in rats treated with pHGF. At 48 h post administration, most hepatocytes had recovered, and not only mitotic hepatocytes (10–13 mitotic cells/100) but also mitotic Kupffer cells were observed. At 72 h, it was evident that the differentiation of hepatic stellate cells (Ito cell) into myofibroblast-like cells and the development of fibrosis around the central veins was prevented by pHGF. These results suggested that (1) pHGF may stabilize cell membranes, (2) pHGF acts as a mitogen not only for hepatocytes but also for Kupffer cells, and (3) pHGF prevents fibrogenesis in the case of CCl4-induced liver injury by preventing the differentiation of hepatic stellate cells.
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  • 5
    ISSN: 1437-773X
    Keywords: Key words Cerebral blood flow ; Blood–brain barrier ; Repeated brief cerebral ischemia ; Hippocampal CA1 ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Neuronal damage and changes in cerebral blood flow (CBF) and the permeability of the blood–brain barrier (BBB) following repeated brief periods of ischemia were studied in Mongolian gerbils. The cerebral ischemia was produced by three repeated occlusions of bilateral common carotid arteries for 3 min at 1-h intervals. CBF and permeability of the BBB were examined with tracers (China ink and silver nitrate) at 1, 3, and 7 days post ischemia using light and electron microscopy. Three days after the reperfusion, significant extravasation of tracers, consequential reduction of CBF, extensive neuronal destruction, and intravascular platelet aggregation were observed. Such vascular changes in the CA1 region were more severe than those in the frontal cortex. These findings strongly support the view that microcirculatory disturbance may be a mechanism responsible for delayed neuronal death in the CA1 region of the hippocampus.
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  • 6
    ISSN: 1432-2307
    Keywords: Key words Inflammation of the lung ; Biopsy ; Ultrastructure ; Laser therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Examination of 127 biopsy specimens from 45 patients with inflammatory lung diseases showed changes consistent with increased permeability of the capillary endothelial cells as an initial stage in the development of the inflammatory reaction. Associated interstitial oedema, deformation of the interalveolar septa, and structural disorganization of alveolar epithelium cells occur, and local microcirculatory problems result in tissue hypoxia and fibrosis. The ultimate morphological picture is determined largely by the intensity of repair. Laser biostimulation minimizes the inflammation and stabilizes fibroplastic process.
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  • 7
    ISSN: 1432-2307
    Keywords: Key words Capillary basement membrane ; Inflammatory myopathy ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The capillary basement membranes from 16 skeletal muscle biopsies from patients with a clinical and histological diagnosis of inflammatory myopathy and from six controls were analysed ultrastructurally and morphometrically. Resin sections from 244 endomysial capillaries were examined by light microscope, and the results were correlated with findings seen in electron micrographs of these capillaries. The ultrastructural morphometric measurements and the statistical analysis showed that the capillary basement membrane was thick and multilaminated in 87% specimens affected by inflammatory myopathy. No thick or multilaminated basement membrane was observed in controls. In inflammatory myopathy the endomysial space next to the capillaries contained an increased amount of collagen fibrils and showed signs of a chronic reparative process. It is suggested that the thick multilaminated basement membrane in inflammatory myopathy represents an advanced stage of vascular regeneration.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Urological research 27 (1999), S. 454-461 
    ISSN: 1434-0879
    Keywords: Key words Cold-restraint stress ; Urinary bladder ; Interstitial cystitis ; Mast cell ; Urothelium ; Ultrastructure ; Ruthenium red ; Flow cytometry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Stress is associated with many diseases of unknown aetiology. This study demonstrates the effects of cold-restraint stress on the morphology of the urinary bladder. Additionally, it compares the results obtained with the morphology of the interstitial cystitis. The animals were subjected to three hours of cold-restraint stress and then starved for 48 h. The morphology and histochemistry of the urinary bladder was investigated with light and electron microscopy. The proliferative activity was analysed via flow cytometry. Increased and degranulated mast cells in the mucosa, leucocyte infiltration in the lamina propria, vacuole formation in the urothelial cells, loose tight junction, dilated intercellular spaces and altered proliferative activity were observed in the stress group when compared with the control. The increase in the number of mast cells and especially degranulated mast cells and vacuole formation and the loose tight junction of the urothelium correlated with the histopathological findings of interstitial cystitis.
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  • 9
    ISSN: 1432-0533
    Keywords: Key words Amyotrophic lateral sclerosis ; Bunina body ; Guam ; Immunohistochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract An investigation of Bunina bodies is important when studying the pathoetiology and pathomechanisms involved in amyotrophic lateral sclerosis (ALS). It may serve as a clue essential for the study of the pathogenesis of Guamanian amyotrophic lateral sclerosis (ALS-G), and it may provide a means of answering the question of whether ALS-G is the same disease as classical ALS or a different entity. In ALS-G, however, no precise histochemical, immunohistochemical, or detailed ultrastructural examination has been published to date. To elucidate the pathological differences/similarities of Bunina bodies between classical ALS and ALS-G, we performed histochemical, immunohistochemical, topographic and ultrastructural examinations. Histochemically, hematoxylin and eosin, Masson’s trichrome, methylgreen-pyronin, phosphotungstic acid-hematoxylin, Klüver-Barrera, Bodian and periodic acid-Schiff staining were utilized. Immunohistochemical examination was performed using antibodies for cystatin C, ubiquitin, Tau-2, Cu/Zn superoxide dismutase, phosphorylated neurofilament and glial fibrillary acidic protein. Histochemical findings were consistent with those previously described for classical ALS. The immunohistochemical study showed that in ALS-G Bunina bodies were intensely labeled by an anti-cystatin C antibody. Topographic examination demonstrated that Bunina bodies were distributed in the spinal anterior horns and Clarke’s column in the spinal cord. Ultrastructurally, Bunina bodies were composed of electron-dense amorphous/ granular material accompanied by vesicular structures and neurofilaments. The results of the present study have revealed that the pathological features of Bunina bodies in ALS-G are identical to those seen in classical ALS. These findings strongly suggest that a similar degenerative process occurs in the spinal anterior horn cells in both ALS-G and classical ALS.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 97 (1999), S. 509-514 
    ISSN: 1432-0533
    Keywords: Key words Hereditary inclusion body myopathy ; Desmin storage myopathy ; Ultrastructure ; Immmunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report two adult familial cases of inclusion body myopathy (IBM) with desmin storage in skeletal muscle. Clinically, both patients presented late-onset, progressive, symmetrical, both proximal and distal muscle weakness. Muscle biopsy findings were identical in both cases and consisted of marked variability in fiber size, increased number of central nuclei and vacuolation involving 10% of fibers. Single or multiple vacuoles were located subsarcolemmally or in the center, and were rimmed by basophilic material. At the ultrastructural level, tubulofilamentous nuclear and cytoplasmic inclusions of 16–21 nm in diameter were frequently observed. In addition, large subsarcolemmal and central deposits composed of electron-dense granular material were present in many fibers. Immunocytochemistry revealed staining for desmin, vimentin and ubiquitin within both inclusions and vacuolated fibers. Possible structural and functional associations between these two types of muscle changes remain unclear. They may either represent two coexistent disease processes or merely reflect an abnormal form of muscle fiber degradation, with unidentifiable specificity.
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 200 (1999), S. 81-90 
    ISSN: 1432-0568
    Keywords: Key words Elastic fibre system ; Microfibrils ; Collagen type IV ; Ultrastructure ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  This study deals with the examination of the elastic fibre system as well as collagen fibrils and collagen type IV in the amnion of the human chorionic plate of uncomplicated pregnancies at term. In organs other than placenta, the elastic fibre system comprises elastic fibres, elaunin and oxytalan microfibrils. The investigation was performed by light and electron microscopy and immunocytochemistry. Abundant oxytalan fibres were present in all amnionic layers, while no elastic fibres were found. Oxytalan microfibrils formed a broad subepithelial layer and were intermingled with collagen fibrils in the subjacent compact layer and in the amnionic mesoderm. Light microscopically, bunches containing orcein-stained oxytalan and collagen-type-IV-immunostained microfibrils were seen rising from the amnionic mesoderm perpendicularly towards the epithelial layer, where they obviously inserted. It can be assumed that the subepithelial microfibrillar layer and the following compact layer form an anchoring zone between the amnionic mesoderm and the epithelium that may contribute to the maintenance of strength. The ultrastructure of the bunches clearly showed collagen fibrils mixed with oxytalan microfibrils. No collagen type I-immunostaining was found in the bunches. After pretreatment of cryostat sections with elastase, oxytalan-orcein-staining was absent, but collagen type IV-immunoreactivity was not altered. Furthermore, after oxytalan-orcein-staining resp. anti-collagen type IV incubation, all positive fibres revealed an identical morphological pattern. We propose that oxytalan and collagen type IV may represent further members of the microfibril complex.
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  • 12
    ISSN: 1432-0533
    Keywords: Key words Ischemia ; Protein synthesis ; Translation ; Ultrastructure ; Hippocampus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract During post-ischemic brain reperfusion there is a substantial reduction of protein synthesis in selectively vulnerable neurons. Normal protein synthesis requires a functional translation initiation complex, a key element of which is eukaryotic initiation factor 2 (eIF2), which in a complex with GTP introduces the met-tRNAi. Phosphorylation of Ser51 on the α subunit of eIF2 [eIF2α(P)] generates a competitive inhibitor of eIF2B, thereby preventing the replenishment of GTP onto eIF2, thus blocking translation initiation. It has been shown that the conditional expression of an eIF2α mutant (Asp substituted for Ser51) imitating the negative charge of Ser51 (P) induces apoptosis. During the first 10 min of post-ischemic reperfusion, there is an approximately 20-fold increase in eIF2α(P) seen in the cytoplasm of CA1 hippocampal neurons, and, by 1 h, there is also accumulation of eIF2α(P) in the nucleus. We utilized post-embedding electron microscopical immunogold methods to examine the localization of eIF2α(P) during reperfusion. Immunogold particles (10 nm) were concentrated chiefly along the rough endoplasmic reticulum and in association with the membranes of the nuclear envelope in CA1 neurons. Aggregations of gold particles in the nucleus were concentrated: (1) within and around the nucleolus, (2) associated to strands of heterochromatin, and (3) along putative nuclear filaments. The presence of eIF2α(P) in the nucleolus probably reflects its association with nascent ribosomal subunits. The β-subunit of eIF2 has a zinc finger and polylysine blocks analogous to those on other proteins that affect transcription. The association of eIF2α(P) with chromatin may have important implications for transcription.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 199 (1999), S. 407-418 
    ISSN: 1432-0568
    Keywords: Key words Placenta ; Vascular wall ; Smooth muscle cell ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The vascular wall structure in the human full-term placental villi of normal pregnancy was studied by means of light and electron microscopy with an improved technique of perfusion fixation and tissue preparation. We observed 81 sections of stem villi that showed cross-sectional profiles of paired vessels in their center. Both vascular walls contained a large amount of extracellular matrix and no elastic lamina between smooth muscle cells of the media, making identification of the artery and the vein quite difficult at first sight. We then noted that the density of the smooth muscle cell population was always considerably higher in one than the other, and identified the former as artery and the latter as vein on the basis of their connection with larger arteries and veins running on the chorionic plate. Between the paired vessels, the artery had a smaller caliber than the vein, and the ratio of venous to arterial caliber was distributed from 1.0 to 2.5. The thickness of media was usually thicker in the vein than in the artery. Clusters of elastic fibers were found occasionally in the media of arteries and veins, and basement membrane-like materials were associated frequently with the elastic fibers and were distributed widely in the media as well as in the adventitia. In the veins, the smooth muscle cells of the most superficial part of the media contained well-developed rough endoplasmic reticulum and Golgi apparatus, indicating differentiation to secrete extracellular matrices. The present study revealed the difference of wall structure between arteries and veins in the placental stem villi for the first time at the ultrastructural level, and suggested differentiation of venous smooth muscle cells, possibly by some influence from the luminal side.
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  • 14
    ISSN: 1432-0533
    Keywords: Key words Polyglucosan body ; Periodic ; acid-Schiff-positive granules ; Mouse brain ; Immunohistochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Periodic acid-Schiff-positive granules (PGs) appear in the mouse brains in relation to advancing age. The exact location and pathophysiological significance of PGs, however, are not fully understood. The incidence, staining properties, and topographical distributions of PGs in the brains of 17 AKR mice ranging in age from 7 to 18 months were examined histochemically and immunohistochemically using antibody KM279 raised against a polyglucosan. In addition, to define the precise site of PG formation, we investigated the brains of 4 AKR mice of 24 months of age using conventional and immunoelectron microscopy. PGs were seen in all mice examined and the levels were increased with age. The PGs were located predominantly in the hippocampus and, to a lesser extent, in the cerebellum and olfactory bulb. Immunohistochemically, PGs in the hippocampus and cerebellum were labeled uniformly with KM279. On immunoelectron microscopy with this monoclonal antibody, the fibrillar or membranous structures corresponding to PGs seen using light microscopy were labeled specifically with gold particles. With conventional electron microscopy, fibrillar or membranous structures were seen along with synaptic vesicles and dense-core granules. Moreover, around the cells containing PGs, a few synaptic junctions with neighboring cells were observed, indicating that the cells contributing to formation of PGs were neuronal cells. The positive immunoreactivity of AKR mouse PGs for the antibody KM279 suggests that the PGs and similar structures in other species may share a common antigenicity. Thus, it is assumed that PGs in AKR mice might result from some abnormalities in glucose metabolism.
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  • 15
    ISSN: 1432-0533
    Keywords: Key words Amyloid precursor protein ; immunoreactivity ; Axonal injury ; Microwave antigen retrieval ; Traumatic brain injury ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Antibodies to the amyloid precursor proteins (APP) have become routine markers for detecting traumatically induced axonal injury (AI) in animals and man. Unfortunately, the techniques used to visualize these proteins are not compatible with routine electron microscopic (EM) analysis. In the current communication, we describe a method for the ultrastructural visualization of antibodies to APP and, using this method, we identify a previously unrecognized population of traumatically injured axons. Rats were subjected to an impact acceleration traumatic brain injury and allowed to survive 30 min to 3 h postinjury. The animals were then perfused, their brains sectioned on a vibratome and the sections prepared for immunocytochemistry using a computer-controlled microwave capable of temperature regulation. The use of temperature-controlled microwave energy unmasked APP antigenic epitopes without sacrificing ultrastructural detail. The APP antibody was found in two distinct populations of reactive axons that differed in size, morphology, location, and temporal progression. Comparable to previous descriptions, one population showed traumatically related reactive changes that led to swelling and disconnection. The other population, however, revealed unanticipated changes reflected in nodal and paranodal swelling of small continuous fibers that showed no evidence of disconnection during the time periods assessed. These studies provide new insight into the complexity of the pathobiology of AI, while describing a novel approach for enhancing APP immunoreactivity at the EM level.
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  • 16
    ISSN: 1432-0568
    Keywords: Key words Intrinsic laryngeal motoneurons ; Cholera toxin HRP ; Ultrastructure ; Swallowing ; Respiration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The laryngeal motoneurons innervating the cricothyroid muscle (CT) are located in the semicompact formation just ventral to the rostral part of the compact formation of the nucleus ambiguus. The motoneurons innervating the posterior cricoarytenoid muscle (PCA) are located in the loose formation. We retrogradely labeled the CT and the PCA motoneurons using cholera toxin subunit B-conjugated horseradish peroxidase, and determined the ultrastructure and synaptic organization of these neurons. The CT and the PCA motoneurons had the appearance of α-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles and a prominent spherical nucleus. Two kinds of neurons were recognized among the PCA motoneurons. The one (PCA-A) was significantly smaller than the other (PCA-B). The average number of axosomatic terminals in a section was significantly largest in the PCA-B (56.6), smaller in the PCA-A (36.0), and smallest in the CT (32.3) neurons. Most of the axosomatic terminals (64.7%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray’s type II) with the PCA-A neurons, while more than 60% contained round vesicles with asymmetric synaptic contacts (Gray’s type I) in the CT (69.5%) and the PCA-B (60.6%) neurons. A few terminals associated with subsurface cisterns were present on all laryngeal motoneurons. These results indicated that the CT motoneurons may receive mostly excitatory terminals, whereas the PCA muscle may be regulated by neurons having many inhibitory terminals, and neurons having many excitatory terminals.
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  • 17
    ISSN: 1432-069X
    Keywords: Key words p75 nerve growth factor receptor ; (p75 NGFr) ; Immunoreactivity ; Ultrastructure ; Prurigo nodularis ; Nerve fiber
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Prurigo nodularis is an inflammatory skin disease characterized by neurohyperplasia. Neurotrophins and their receptors play a critical role in nerve growth, differentiation, maturation and maintenance, including cutaneous nerve fiber growth and innervation. They may also be responsible for events related to the growth and differentiation control of keratinocytes. To explore the exact distribution of the p75 low-affinity nerve growth factor receptor (p75 NGFr) in the cutaneous nerve components, p75 NGFr immunofluorescence as well as ultrastructural immunohistochemical studies were performed on prurigo nodularis lesional skin and normal human skin samples. The immunofluorescence results revealed that nerve fibers and bundles were increased in number and size in lesional upper dermis with stronger p75 NGFr immunoreactivity than in the corresponding normal tissue. At the ultrastructural level, a lot of nerve fibers clustered together in the prurigo nodularis dermal tissue. The axons were enlarged and branched, but the axons themselves seldom showed any NGFr immunoreactivity. The Schwann cell bodies were extended and irregularly shaped, and tended to separate into many branches enveloping the axons. The Schwann cell membrane showed strong p75 NGFr immunoreactivity. The perineurium cells also revealed strong p75 NGFr immunoreactivity. The Schwann cells inside the perineurium were less p75 NGFr-immunoreactive than those outside the perineurium. The membrane of certain basal keratinocytes showed NGFr immunoreactivity as well. The present results indicate that overexpression of p75 NGFr in Schwann cells and perineurium cells could contribute to the neurohyperplasia in prurigo nodularis.
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  • 18
    ISSN: 1432-041X
    Keywords: Key words Imaginal disc ; Axonal trajectories ; Ultrastructure ; Chaoborus (Insecta ; Diptera)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  In one of his classical studies on insect metamorphosis, Weismann compared the imaginal anlagen of the ancestral phantom midge, Chaoborus, with those of advanced brachycerans. We have expanded his findings on the relationships between larval and imaginal organs using electron microscopy and cobalt backfilling of the antenna and leg anlagen and the axonal trajectories of corresponding larval sensilla. We show that both primordia are confluent with the larval antennae and ”leg” sensilla (an ancestral Keilin organ), respectively. These fully developed larval organs represent the distal tips of the imaginal anlagen rather than separate cell clusters. The axons of the larval antenna and leg sensilla project across the corresponding anlagen to their target neuromeres within the central nervous system (CNS). Within the discs, nerves composed of these larval axons, developing afferent fibres and efferences ascending from the CNS are found. Both the structure of the primordia and the axonal trajectories thus relate the situation found in advanced brachycerans with that seen in more ancestral insects. In addition, the larval antennae, legs, wings and even the eyes possess very similar afferent pioneer trajectories supporting the idea that the described pattern is generally used in the ontogeny of sensory systems.
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  • 19
    Electronic Resource
    Electronic Resource
    Springer
    Sexual plant reproduction 12 (1999), S. 99-109 
    ISSN: 1432-2145
    Keywords: Key words Arabidopsis thaliana ; Megasporogenesis ; Meiosis ; Ultrastructure ; Cellular polarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  In this study, megasporogenesis of the plant model Arabidopsis thaliana was investigated by electron microscopy for the first time. The data described here could constitute a reference for future investigations of Arabidopsis mutants. During the beginning of meiosis the megaspore mother cell shows a polarity created by unequal distribution of organelles in the cytoplasm. Plastids accumulate in the chalazal region and long parallel saccules of endoplasmic reticulum, small vacuoles and some dictyosomes are found in the micropylar region. Plasmodesmata are abundant in the chalazal cell wall. The nucleus is almost centrally localized and contains a prominent excentric nucleolus and numerous typical synaptonemal complexes. After the second division of meiosis the four megaspores are separated by thin cell walls crossed by numerous plasmodesmata and do not show significant cellular organization. The young functional megaspore is characterized by a large nucleus and a large granular nucleolus. The cytoplasm is very electron dense due to the abundance of free ribosomes and contains the following randomly distributed organelles: mitochondria, a few short saccules of endoplasmic reticulum, dictyosomes and undifferentiated plastids. However, there is no apparent polarity, except for the distribution of some small vacuoles which are more abundant in the micropylar region of the cell. The degenerating megaspores are extremely electron dense and do not show any substructure.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Experimental brain research 124 (1999), S. 89-99 
    ISSN: 1432-1106
    Keywords: Key words Microglial culture ; Brain macrophages ; Isolectin ; Ultrastructure ; Intracellular pathway
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The present study examined the lectin labeling of diverse morphological forms of microglia in culture. Similar to amoeboid microglial cells in vivo, polymorphic microglia showed lectin labeling at their plasma membranes, as well as in a few cytoplasmic vesicles and vacuoles. This labeling pattern was observed in cultured microglia incubated with isolectin at 4°C for 30 min. Five minutes after the temperature was raised to 37°C, the surface lectin receptors appeared to be internalized, as shown by the occurrence of many subsurface lectin-labeled vesicles, vacuoles and tubule-like structures. With longer incubation (up to 1–2 h at 37°C), many lysosomes and a few trans-Golgi saccules and associated lysosome-like structures became labeled. Concomitant with these changes was a reduction of lectin labeling at the plasma, with labeling having vanished in most of the cells after 1–2 h of incubation. By 24 h, only a few cells retained surface lectin labeling. It appears, therefore, that irrespective of morphology, lectin labeling (including its intracellular pathway) of microglia in culture parallels that of amoeboid microglia in vivo. This would offer a useful model for the study of lectin turnover in microglia and help to explain the roles of such receptors in microglial differentiation and function.
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  • 21
    ISSN: 1432-072X
    Keywords: Key wordsClostridium xylanolyticum ; Cinnamic acid ; Esterase ; Lignocellulose ; Sporogenesis ; Ultrastructure ; Cell envelope
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.
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  • 22
    ISSN: 1432-0878
    Keywords: Key words Chondrocyte ; Synoviocyte ; Co-culture ; Proliferation ; Lipid peroxidation ; Cytotoxicity ; Ultrastructure ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Objective: A new co-culture system of rat articular chondrocytes and synoviocytes (HIG-82; cell line) was incubated with phorbol myristate acetate (PMA), H2O2 or a combination of Fe2+ and ascorbic acid to simulate inflammation-like radical attacks in articular joints. Methods: Chondrocytes were characterized by immunocytochemistry against collagen type II, transmission electron (TEM) and light microscopy. Lipid peroxidation was investigated by measuring thiobarbituric-acid-reactive material in the supernatants, cytotoxicity by determining release of lactate dehydrogenase and proliferation by measuring [3H]thymidine incorporation, culture protein and DNA. Results: PMA or Fe2+ and ascorbic acid induced lipid peroxidation in chondrocytes and synoviocytes that was decreased significantly in co-cultures. PMA and H2O2 dose dependently induced release of lactate dehydrogenase in chondrocytes, which was lowered in co-cultures or in previously co-cultured chondrocytes to a nearly basal level. In contrast, conditioned media of synoviocyte cultures showed no lowering effect on the radical-induced toxicity. Protection against H2O2-induced damage of cellular membranes by co-culturing was also shown by TEM. Synoviocytes released chondrocyte-stimulating growth factors spontaneously without previous interaction. Conclusion: Chondrocytes establish protective mechanisms against reactive oxygen species via an interaction with synoviocytes. Our co-culture model presents a possible way to study mechanisms of inflammation in articular joints under defined conditions.
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  • 23
    ISSN: 1432-0878
    Keywords: Key words Chloride cells (mitochondria-rich cells) ; Teleost larvae ; Osmoregulation ; Immunohistochemistry ; Quantification ; Ultrastructure ; Oreochromis mossambicus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Integumental and branchial chloride cells of tilapia larvae (Oreochromis mossambicus) were studied at the light-microscopical and ultrastructural level. Total numbers and distribution of chloride cells were quantified after immunostaining of cross sections of the entire larvae with an antibody against the α-subunit of Na+/K+-ATPase. The majority (66%) of Na+/K+-ATPase-immunoreactive (ir) cells, i.e. chloride cells, of freshwater tilapia larvae were located extrabranchially up to 48 h after hatching. Five days after hatching, the majority (80%) of chloride cells were found in the buccal cavity. Transfer of 24-h-old larvae to 20% sea water speeded up this process; 24 h after transfer (i.e. 48 h after hatching), the majority (59%) of chloride cells were located in the buccal cavity. The branchial chloride cell population of 24-h- and 120-h-old larvae consisted of immature, mature, apoptotic and necrotic chloride cells. However, relatively more immature chloride cells were observed in freshwater larvae (42–63%) than in (previously studied) freshwater adults (21%), illustrating the developmental state of the gills. After transfer to sea water, the incidence of degenerative chloride cells did not change. Furthermore, the incidence of immature cells had decreased and a new subtype of chloride cells, the ”mitochondria-poor” cells, appeared more frequently. These mitochondria-poor chloride cells were characterised by an abundant tubular system and relatively few mitochondria, which were aligned at the border or concentrated in one part of the cytoplasm. Most of these cells did not contact the water. The function of their enhanced appearance after seawater transfer is unknown.
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  • 24
    ISSN: 1432-0878
    Keywords: Key words Mechanoreceptors ; Synaptic proteins ; Histochemistry ; Ultrastructure ; Slit sensilla ; Hair sensilla ; Cupiennius salei (Chelicerata)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7–8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5–6.8 μm long and located 0.5–6.6 μm apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results.Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.
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  • 25
    ISSN: 1432-0878
    Keywords: Key words Teleost fish ; Puberty ; Testes ; Sex steroids ; Ultrastructure ; Steroidogenesis ; Clarias gariepinus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The present report focuses on the mechanism(s) involved in the steroid-induced decrease of androgen production in immature African catfish testes that was observed in previous studies. Juvenile animals were implanted with Silastic pellets containing different 11-oxygenated androgens (11-ketotestosterone, KT; 11β- hydroxyandrostenedione, OHA; 11-ketoandrostenedione, KA), testosterone (T) or estradiol-17β (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were either incubated with increasing concentrations of catfish luteinizing hormone (LH), or incubated with [3H]-pregnenolone ([3H]-P5) or [3H]-androstenedione ([3H]-A). Tissue fragments were also prepared for the quantitative assessment of Leydig cell morphology. Most of the parameters studied were not affected significantly by implantation of E2. Implantation of all androgens inhibited both the basal and the LH-stimulated androgen secretory capacity in vitro. This was associated with a reduced size of the Leydig cells and loss of half of their mitochondria. The studies on the metabolism of tritiated steroid hormones indicated that steroidogenic steps prior to 11β-hydroxylation, probably C17–20 lyase activity, were affected by all androgens. Although the effects of 11-oxygenated androgens and T on Leydig cells were mostly similar, previous work showed that only the 11-oxygenated androgens stimulated spermatogenesis, suggesting that distinct mechanisms of action are used by 11-oxygenated androgens and T. These mechanisms, however, seem to merge on the same target(s) to impair Leydig cell androgen production. Such a negative feedback mechanism may be of relevance in the context of the decline in androgen secretion per milligram testis tissue that accompanies the first wave of spermatogenesis in pubertal African catfish.
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  • 26
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 295 (1999), S. 151-158 
    ISSN: 1432-0878
    Keywords: Key words Class II MHC-positive cells ; Human leukocyte antigen-DR ; Dental pulp ; Dendritic cells ; Macrophages ; Ultrastructure ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)-DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.
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  • 27
    ISSN: 1432-0878
    Keywords: Key words Neuromelanin ; Neuron ; Peroxidase ; Oxygen metabolism ; High-definition light microscopy ; Electron microscopy ; Ultrastructure ; Cytochemistry ; Substantia nigra ; Lumbricusterrestris (Annelida)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Histochemical examination of 1-μm tissue sections from the dorsal nerve plexus of the earthworm, Lumbricus terrestris, reveals multiple brown intraneuronal granules. These granules contain material morphologically and histochemically consistent with neuromelanin. When viewed with transmission electron microscopy, these were seen as single membrane-enclosed biphasic granules with diameters of 370–730 nm. Exposure of L. terrestris to high-level environmental oxygen resulted in an increase in the number of neuromelanin-like pigment granules within the neurons of the circular muscle layer. As measured by ortho-phenylenediamine hydrochloride, the endogenous peroxidase activity of extracts from worms incubated in high-level environmental oxygen was 51% more than controls. The endogenous peroxidase activity was localized in situ with 3,3-diaminobenzidine (DAB) and was found to increase in and around the neuromelanin-like pigment-containing neurons within the circular muscle layer. These studies suggest that the nerve net of L. terrestris may serve as a model to study the role of neuromelanin production in oxidative stress and its relationship to endogenous peroxidases.
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  • 28
    ISSN: 1432-0878
    Keywords: Key words Melanin-concentrating hormone neurons ; Lateral hypothalamic slice culture ; Immunocytochemistry ; Ultrastructure ; In situ hybridization ; Competitive RT-PCR ; Leptin assay ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Hypothalamic slices containing the lateral hypothalamic area (LHA) were prepared from 6- to 8-day-old rats and maintained in stationary culture for up to 35 days in order to analyse how well the melanin-concentrating hormone (MCH) neurons survived. As previously reported for other brain areas, this method yielded a long-term well-preserved organotypic organization. Light- and electron-microscopic investigations showed that differentiation continued and that synaptic contacts developed in vitro. After a period of elimination of damaged cells and fibres, most of the remaining neurons and glial cells retained a normal morphology throughout the culture period. MCH neurons, in particular, survived well as attested by the strong immunocytochemical and in situ hybridization signals still observed after several weeks. In a comparison with the day of explantation, competitive reverse transcription/polymerase chain reaction demonstrated the remarkable stability of the level of MCH mRNA at least until the 20th day in culture; after 30 days, the clear decrease in this level seemed to be correlated with a loss of MCH neurons, rather than with a decrease in MCH expression. After 10 days of culture, the incubation of slices in the presence of the hormone leptin (50 ng/ml) resulted in a strong decrease of MCH gene expression, suggesting that MCH neurons retained their physiological properties. Thus, the LHA slice stationary culture, especially between one and three weeks (i.e. after tissue stabilization and before extensive cell loss), appears to be a suitable method for physiological and pharmacological studies of these neurons.
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  • 29
    ISSN: 1432-0878
    Keywords: Key words ECL cells ; Gastrin ; Reserpine ; Organelles ; Ultrastructure ; Rat (Sprague-Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The histamine-storing ECL cells in the stomach play a key role in the control of acid secretion. They contain granules, secretory vesicles and microvesicles, and sustained gastrin stimulation results in the additional formation of vacuoles and lipofuscin bodies. The cells are rich in the vesicle monoamine transporter type-2 (VMAT-2), which can be inhibited by reserpine. The present study examines the effect of reserpine on ECL-cell ultrastructure and histamine compartmentalization. Rats received reserpine and/or gastrin. Reserpine was given twice by the intraperitoneal route (25 mg/kg once daily). Gastrin-17 was given by subcutaneous infusion (5 nmol/kg/h), starting at the time of the first reserpine injection and continuing for 4 days when the rats were killed. At this stage, histamine in the oxyntic mucosa was unaffected by reserpine but elevated by gastrin. Immunocytochemical analysis (confocal microscopy) showed ECL-cell histamine in control and gastrin-treated rats to be localized in cytoplasmic organelles (e.g., secretory vesicles). After treatment with reserpine alone or reserpine+gastrin, ECL-cell histamine occurred mainly in the cytosol. Planimetric analysis (electron microscopy) of ECL cells showed reserpine to increase the number, size and volume density of the granules and to reduce the size and volume density of the secretory vesicles. Gastrin reduced the number and volume density of granules and secretory vesicles, increased the number and volume density of microvesicles and caused vacuoles and lipofuscin bodies to appear. Reserpine+gastrin increased the number, volume density and size of the granules. Reserpine prevented the effects of gastrin on secretory vesicles, vacuoles and microvesicles, but did not prevent the development of lipofuscin. Our findings are in line with the views: (1) that preformed cytosolic histamine is taken up by granules/secretory vesicles via VMAT-2, that histamine is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement and (2) that vacuoles are formed by the fusion of large secretory vesicles.
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  • 30
    ISSN: 1615-6102
    Keywords: Cumulus oophorus ; Ovarian follicle ; Fertilization ; Ultrastructure ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The fine structure of the human cumulus oophorus has been reviewed on the basis of scanning and transmission electron microscopic observations as well as of immunofluorescence data. Tissues sampled from preovulatory ovarian follicles and cumulus-enclosed oocytes and fertilized eggs (collected from the oviduct or obtained during in vitro fertilization procedures) have been evaluated from a microtopographic and morphodynamic point of view in order to better clarify the possible role of this population of cells. In particular, the following aspects have been studied and discussed: the presence of multiple close contacts (modulated by the interposition of the zona pellucida) between the oocyte surface and the long microvillous evaginations projecting from the inner aspect of corona cells surface (through these structures the intraovarian cumulus oophorus may control oocyte growth and metabolism up until the time of ovulation); the occurrence of different subpopulations of cells (steroid-synthetic cells, cells producing adhesive proteins, leukocytes, macrophages) in the postovulatory, extraovarian cumulus oophorus surrounding oocytes, zygotes and early developing embryos. All these elements found in the cumulus mass may positively act, through their paracrine activities, on the chemical composition of the microenvironment in which fertilization occurs.
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  • 31
    ISSN: 1615-6102
    Keywords: Plasmalemmic cord ; Pollen grain ; Ultrastructure ; Magnolia ×soulangeana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary InMagnolia ×soulangeana pollen grains the generative cell (GC) does not become totally free within the vegetative cell (VC), at least until the pollen tube emergence. Due to a deviation in its detachment process from the sporoderm, the opposing ends of the VC plasmalemma do not fuse themselves when the GC moves away from the intine. Consequently, the interplasmalemmic space surrounding the GC does not become isolated but rather maintains continuity with the sporoderm through a complex formation that we have called plasmalemmic cord. The real existence of this formation was confirmed through serial sectioning showing the plasmalemmic cord to consist of the VC plasmalemma. In its initial portion it is occupied by a reasonably accentuated wall ingrowth of the inner layer of the intine (intine 3). In the remainder portion, neither of the cytochemical tests used in this work have revealed the presence of a significant amount of wall material. However, ultrathin sections of samples processed either chemically or by cryofixation showed the existence of an intricate system of tubules and vesicles, some of which are evaginations of the VC plasmalemma. The hypothesis that the plasmalemmic cord may have a role in the complex interactions between the two pollen cells is discussed.
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  • 32
    ISSN: 1615-6102
    Keywords: Anatomy ; Floral nectary ; Modified stomata ; Phloem ; Pisum sativum ; Stereology ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The floral nectary ofPisum sativum L. is situated on the receptacle at the base of the gynoecium. The gland receives phloem alone which departed the vascular bundles supplying the staminal column. Throughout the nectary, only the companion cells of the phloem exhibited wall ingrowths typical of transfer cells. Modified stomata on the nectary surface served as exits for nectar, but stomatal pores developed well before the commencement of secretion. Furthermore, stomatal pores on the nectary usually closed by occlusion, not by guard-cell movements. Pore occlusion was detected most frequently in post-secretory and secretory glands, and less commonly in pre-secretory nectaries. A quantitative stereological study revealed few changes in nectary fine structure between buds, flowers secreting nectar, and post-secretory flowers. Dissolution of abundant starch grains in plastids of subepidermal secretory cells when secretion commenced suggests that starch is a precursor of nectar carbohydrate production. Throughout nectary development, mitochondria were consistently the most plentiful organelle in both epidermal and subepidermal cells, and in addition to the relative paucity of dictyosomes, endoplasmic reticulum, and their associated vesicles, the evidence suggests that floral nectar secretion inP. sativum is an energy-requiring (eccrine) process, rather that granulocrine.
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  • 33
    ISSN: 1615-2573
    Keywords: Endomyocardial biopsy ; Dilated cardiomyopathy ; Children ; Ultrastructure ; Basal lamina layering of capillary
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We analyzed the electron-microscopic features of endomyocardial biopsy from pediatric patients with dilated cardiomyopathy (DCM). The specimens examined were taken from the right ventricle of ten patients aged from 2 to 15 years (mean 9.7 years). Biopsy specimens from eight patients with congenital heart disease (tetralogy of Fallot), aged from 3 to 12 (mean 7.3 years), and ten adult patients with DCM, aged from 32 to 60 (mean 45 years), were also examined. Patients considered to have endocardial fibroelastosis, arrhythmogenic right ventricular cardiomyopathy, specific cardiomyopathy, or coronary heart disease were excluded from this study. Specimens from pediatric patients with DCM showed various degrees of ultrastructural abnormalities of myocytes, including myofibrillar fragmentation, mitochondrial abnormalities, and intracellular edema. The ultrastructurally determined contractility failure index based on the severity of myocardial degeneration at the electronmicroscopic level was 4.9 ± 1.1. This value was significantly higher than that in patients with tetralogy of Fallot (0.9 ± 0.6,P 〈 0.001) but was not significantly different from that in adult patients with DCM (6.1 ± 2.6). The index of pediatric patients with DCM who died within 3 years was high (6.0 ± 0.8). Basal lamina layering of a capillary (BLL) in the myocardium was revealed in 1 of the 10 (10%) pediatric patients with DCM and in 6 of the 10 (60%) adult patients with DCM (P 〈 0.05). No BLL was noted in the patients with tetralogy of Fallot. These findings may be related to the pathogenesis of DCM in children and adults.
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  • 34
    ISSN: 1615-6102
    Keywords: Endosymbiont ; Mycetocyte ; Mycetome ; Oocyte ; Transovarial transmission ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary All anoplurans live symbiotically with prokaryotic microorganisms hosted in specialized cells, termed mycetocytes. In nymphs and males mycetocytes are distributed between midgut epithelial cells. In females, besides the midgut, mycetocytes are found in the reproductive organs where they are located at the base of ovarioles in contact with lateral oviducts. The mycetocyte-associated symbionts are transmitted from one generation to the next transovarially. Here, the results of histological and ultrastructural studies on the distribution and transmission of symbiotic microorganisms within the ovaries of the anopluranHaematopinus suis are presented. Interestingly, during advanced oogenesis (i.e., choriogenesis) of this species all symbionts are localized extracellularly and form a tight mass located at the posterior pole of the oocyte just below the hydropyle. In insects studied so far, such localization of transovarially transmitted microorganisms has been reported only in the closely related speciesHaematopinus eurysternus.
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  • 35
    ISSN: 1432-136X
    Keywords: Key words Freeze tolerance ; Sciatic nerve ; Cryoinjury ; Dehydration ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells.
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  • 36
    Electronic Resource
    Electronic Resource
    Springer
    Parasitology research 85 (1999), S. 999-1006 
    ISSN: 1432-1955
    Keywords: Key wordsEchinococcus granulosus ; Praziquantel ; Metacestode ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The efficacy of praziquantel against the metacestode of Echinococcus granulosus was studied by means of in vitro incubations or in vivo experiments. The results of in vitro incubations indicated that the effectiveness of praziquantel was higher when the parasite material comprised cysts from cyst masses than in the case of intact cysts that retained their adventitial layer. Ultrastructural alterations in the germinal layer of collapsed cysts incubated in vitro were detected. The results obtained in mice after 4 months of treatment demonstrated no significant difference between the control and treated groups with regard to the number and wet weight of developed cysts. However, ultrastructural alterations were detected in the cyst tissue that were similar to those described in the in vitro experiment. In contrast, the effect of chemoprophylaxis on the number and the wet weight of developed cysts was extremely significant as compared with the control value, the efficacy being 99.41% and 98.32%, respectively. Moreover, ultrastructural observations of the cyst tissue revealed loss of its integrity, and no intact cyton was observed in the germinal layer of the developed cyst.
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  • 37
    ISSN: 1432-1955
    Keywords: Key words Monogenea ; Capsalidae ; Benedenia rohdei ; B. lutjani ; Ectoparasites ; Lutjanus carponotatus ; Glands ; Ultrastructure ; Adhesion ; Attachment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The anterior adhesive areas of Benedenia rohdei from the gills and B. lutjani from the pelvic fins of Lutjanuscarponotatus at Heron Island, Australia, were studied using scanning and transmission electron microscopy. All specimens were fixed when detached from host tissue. Both monogenean species have two disc-like anteroventral attachment organs, each of which has an anterolateral adhesive area divided into three adjacent zones by tegument from the ventral surface of the attachment organ. A rod-shaped secretion and a smaller, roughly spherical secretion are associated with the anterior adhesive areas in both species; a third type of secretion occurs anteriorly but outside these adhesive areas. The electron-dense spherical secretory bodies released onto the anterior adhesive zones in these Benedenia spp. are of a single type and differ ultrastructurally from those previously reported in monogeneans living on teleost hosts. A correlation, therefore, between secretion morphology and host type is not supported. No relationship was found between parasite microhabitat and secretion morphology.
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  • 38
    ISSN: 1861-387X
    Keywords: Meningioma ; Intranuclear inclusion ; Immunohistochemistry ; Ultrastructure ; Intermediate filament
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We describe a case of meningothelial meningioma with a large number of intranuclear inclusions. Morphologically, these are divided into cytoplasmic inclusions and nuclear vacuoles. The cytoplasmic inclusion has a limiting membrane with cell organelles and filaments. Inclusions of this type are generally eosinophilic, like the cytoplasm. However, there are many inclusions that are more eosinophilic than the cytoplasm or that have a ground-glass appearance. Some of them may contain fine or coarse granules. On the other hand, the nuclear vacuole lacks a limiting membrane and appears empty. In most of the inclusions of this type, there is a faintly basophilic substance in the margin. Generally, the cytoplasmic inclusions are as immunopositive as cytoplasm with vimentin, but some of these cytoplasmic inclusions are more reactive. Under the electron microscope, abnormal aggregation of intermediate filaments is recognized in the cytoplasmic inclusions. It is considered that a strong reaction of cytoplasmic inclusions with vimentin immunostaining is due to abnormal aggregation of intermediate filaments. The present study distinctly demonstrates abnormal localization of intermediate filaments in the cytoplasmic inclusions, and it is suggested that the cytoskeleton participates in the evolution of the cytoplasmic inclusions.
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  • 39
    ISSN: 1432-2307
    Keywords: Key words Apoptosis ; Crescent-shaped spaces ; Ultrastructure ; Nivalenol ; Thymus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  There have been many reports on the formation of apoptotic bodies, but little is known about the cellular pathological processes and the morphological changes involved. We induced apoptotic cell death by administering nivalenol (NIV), a trichothecene mycotoxin produced by Fusarium species, and investigated the ultrastructural process of formation of apoptotic bodies. The thymus was examined by electron microscopy 6, 12, and 18 h after administration. Apoptotic cell death was induced in the thymus of NIV-treated mice. The nuclei became invaginated and pinched off to give fragments, and crescent-shaped spaces (CSS) were found around the nuclear envelopes of these cells at quite an early stage. In some of these spaces, myelin figures were observed. We divided the process of formation into four stages and characterized each of them. These are easily recognized in morphological stages and are also useful for clarifying the apoptotic mechanism.
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  • 40
    ISSN: 1432-0533
    Keywords: Key words Brittany Spaniel dog ; Immunocytochemistry ; Purkinje cell ; Spinocerebellar degeneration ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Eight Brittany Spaniel dogs, seven females and one male, between 7 and 14 years old presented with clinical neurological signs of spinocerebellar disease of about 6 months to 4 years duration. Clinically the dogs had a dramatic forward “saluting” movement of the thoracic limbs, hypermetria of the pelvic limbs, cerebellar ataxia and intention tremors. Terminally, dogs crawled in a crouched thoracic posture with neck extension. Lesions were confined to cerebellum, medulla oblongata and spinal cord. The most severe lesion was diffuse Purkinje cell loss with massive neurofilament accumulation in degenerating cells. There was some bilateral neuronal degeneration in the dorsal horns of the spinal cord and in the gracilis and cuneate nuclei. There was bilateral sporadic axonal degeneration in the dorsal columns and lateral and ventromedial areas of the spinal cord. The etiology of this syndrome was not determined.
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  • 41
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    Acta neuropathologica 95 (1998), S. 367-372 
    ISSN: 1432-0533
    Keywords: Key words Amyotrophic lateral sclerosis ; Heterotopic neuron ; Alpha motor neuron ; Immunocytochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract This report concerns a comparative immunocytochemical, ultrastructural and morphometric investigation on heterotopic neurons in the white matter of the spinal cords of 19 patients with amyotrophic lateral sclerosis (ALS) and 18 age-matched neurologically normal individuals. The study revealed that the heterotopic neurons were scattered in the white matter, often adjacent to gray matter, that they immunoreacted with the antibody to synaptophysin, and that there were synaptic apparatuses on the surface of their somata and their neuronal processes. Bunina bodies and ubiquitin-positive inclusions such as Lewy body-like inclusions and skein-like inclusions, characteristic of anterior horn neurons of ALS, were present in the cytoplasm of the patients’ heterotopic neurons in the anterior or lateral column of the white matter. These findings suggest that heterotopic neurons in the anterior or lateral column have the characteristics of alpha motor neurons. The average number of heterotopic neurons observed in ALS patients was generally less than in normal subjects. This reduction was correlated with the severity of neuronal loss. The heterotopic neurons in ALS were less susceptible to the degenerative process as compared with spinal cord anterior horn cells. We assume that in this disease the heterotopic neurons may be degenerated and their number diminished after or concomitantly with the depletion of anterior horn neurons.
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  • 42
    ISSN: 1432-0533
    Keywords: Key words Dentatorubral-pallidoluysian atrophy ; Nuclear inclusion ; Ubiquitin ; Immunohistochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We examined the brains and spinal cords of seven patients with clinicopathologically and genetically confirmed hereditary dentatorubral-pallidoluysian atrophy (DRPLA) using an antibody against ubiquitin, and found small, round immunoreactive intranuclear inclusions in both neurons and glial cells in various brain regions. Ubiquitinated neuronal intranuclear inclusions (uNIIs) were consistently found in the striatum, the pontine nuclei, the inferior olivary complex, the cerebellar cortex and the dentate nucleus. Ubiquitinated glial intranuclear inclusions (uGIIs) were found less frequently than uNIIs. Most of the inclusion-bearing nuclei were of an astrocytic nature. Immunostaining with an antibody against DRPLA protein revealed similar immunoreactive neuronal and glial intranuclear inclusions, but in much smaller in numbers compared with uNIIs and uGIIs. Electron microscopy showed that such inclusions were composed of granular and filamentous structures. These findings strongly suggest that, in DRPLA, the occurrence of uNIIs and uGIIs is directly related to the causative gene abnormality (an expanded CAG repeat encoding polyglutamine), that neurons are affected much more widely than previously recognized and that glial cells are also involved in the disease process.
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  • 43
    ISSN: 1432-0533
    Keywords: Key words Congenital myopathy ; Muscle fibers ; Ultrastructure ; Myofibrillar disarray
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A 44-year-old man presenting with dyspnoic attacks was found to be affected with congenital myopathy, rigid spine, restrictive respiratory insufficiency and cardiomyopathy. Muscle biopsy showed type 1 fiber predominance (65.7%) and hypotrophy, and characteristic changes in 43.9% of the type 1 fibers, consisting in alternating pale and dark staining on alkaline ATPase reacted sections in a mosaic pattern. Ultrastructural examination demonstrated bands of myofibrils at right angles or skew to the remaining myofibrils transversing the fibers. Myofibrillar disarray was always associated with loss of the Z-discs and actin filaments, and often with aggregation of mitochondria. The muscle biopsy findings in this patient suggest a new entity of congenital myopathy with clinical features of rigid spine, cardiomyopathy and restrictive respiratory insufficiency, characterized by peculiar abnormalities of ATPase staining in a mosaic pattern and, ultrastructurally, by zones of disorientation of the sarcomeres.
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  • 44
    ISSN: 1432-0568
    Keywords: Key words Adult-type Leydig cells ; Endothelium ; 3β-HSD ; Ultrastructure ; Differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In the biosynthesis of steroid hormones 3β-hydroxysteroid dehydrogenase (3β-HSD) is a key enzyme. The present report describes the subcellular localization of the enzyme in the fetal-type Leydig cells, the fibroblast-like precursors of adult-type Leydig cells and in endothelial cells of interstitial capillaries. Histochemical methods for light microscopy and ultracytochemical methods for electron microscopy were used on rat testes of postnatal day 15. 3β-HSD reactivity was located at subcellular levels by means of the ferricyanide method. A specific, distinct localization of reaction product in the form of copper ferrocyanide precipitates was observed on the membranes of the smooth endoplasmic reticulum not only in the fetal-type Leydig cells and the fibroblast-like precursors of adult-type Leydig cells, but also focally in the endothelial cells of interstitial blood capillaries. Topographically, the 3β-HSD-positive precursors were most often found in the outer layer of the boundary tissue and surrounding interstitial blood vessels. The capillaries with 3β-HSD-positive endothelial cells were usually located in the vicinity of 3β-HSD-positive Leydig cells. For the first time, 3β-HSD has been located at the subcellular level in precursors of adult-type Leydig cells and focally in capillary endothelial cells associated with them. Due to the close association between 3β-HSD-positive vascular endothelial cells and Leydig cells a paracrine relationship between the two cell types may be involved in the acute regulation of steroidogenesis by blood-borne luteinizing hormone.
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  • 45
    ISSN: 1432-0533
    Keywords: Key words Anorexia nervosa ; Myopathy ; Muscle biopsy ; Ultrastructure ; Protein-energy malnutrition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Protein-energy malnutrition in anorexia nervosa is an under-recognised cause of muscle dysfunction. To characterise the skeletal myopathy that occurs in patients with severe anorexia nervosa, muscle function and structure were comprehensively examined in eight young adult female patients with severe (40%) self-induced weight loss. All of the patients showed impaired muscle function on strength and exercise measurement. The maximum voluntary contraction force for the patient group was significantly less than predicted values. Electromyography revealed myopathy in five of the patients, four of whom also had electro-physiological evidence of neuropathy. However, muscle biopsy specimens consistently showed myopathic changes with severe type 2 fibre atrophy but with no evidence of neuropathic changes. Ultrastructurally, there was separation and segmental loss of myofibrils and most biopsy samples contained abundant glycogen granules; we have previously reported that one of the most consistent biochemical abnormalities in these patients is impaired ischaemic lactate responses to forearm exercise. The result of severe protein-energy malnutrition on the musculo-skeletal system is a metabolic myopathy. Although the patients admitted to a variety of abnormal dieting behaviours, such as over-exercising and self-induced vomiting, no association was found between any of these and quantitative histological changes in the muscle biopsy samples. It is recommended that myopathy in anorexia nervosa be treated by instituting an appropriate refeeding programme.
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  • 46
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    Anatomy and embryology 197 (1998), S. 135-141 
    ISSN: 1432-0568
    Keywords: Key words Interstitial cells of Cajal ; Ultrastructure ; Gap junction ; Intestine ; Motility
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Interstitial cells forming many large gap junctions in the region of the deep muscular plexus of the rat small intestine were studied by electron microscopy and by three-dimensional cell models reconstructed from serial ultrathin sections. Two different profiles of cells were observed. Cells of the first profile are characterized by an elongated cell shape and by less electron-dense cytoplasm, containing many mitochondria, well-developed Golgi apparatus and free ribosomes. They mainly connect with smooth muscle cells of the main circular layer. In a three-dimensional cell model, the total area of the gap junctions occupies 1.3% of the cell surface. Cells of the second profile are characterized by the frequent occurrence of slender cytoplasmic processes, higher electron-dense cytoplasm, containing mitochondria, Golgie apparatus and well-developed rough endoplasmic reticulum, and numerous caveolae on the cell membrane. In this cell model, gap junctions occupy 0.8% of the cell surface. The ratio of gap junctions with the same profile of cells to the total gap junction area is 37.7%, which is more than three times greater than the 9.9% in cells of the first profile. These cells were closely associated with nerve terminals. It is likely that these cells with different profiles constitute subtypes with each other and cooperate for regulation of intestinal motility via the transmission of nerve signals.
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  • 47
    ISSN: 1432-0533
    Keywords: Key words Congenital myopathy ; morphometry ; Ultrastructure ; Fetal myogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Centronuclear myopathy (CNM) is a congenital myopathy which manifests itself as a severe neonatal (also termed myotubular myopathy), early-onset, or adult form. The histological pattern of each is marked by a considerable number of nuclei of muscle fibers being internally placed. Owing to their remote resemblance to myotubes, and their expression of developmentally regulated proteins, most authors now favor the concept that myogenesis is arrested or delayed in this disease. We here present two muscle biopsy specimens of a patient with early-onset CNM, taken at the age of 5 months and 14 years, respectively. The first biopsy sample contained internally placed nuclei in 7% of the muscle fibers, abundant minute myotubes, and hypertrophic muscle fibers. The second biopsy sample showed internally placed nuclei in 40% of the muscle fibers, and hypotrophic fibers. We suggest that the histological findings in early-onset CNM are the result of a complex dynamic process, which includes a delay in maturation.
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  • 48
    ISSN: 1432-0533
    Keywords: Key words Aging ; Blood-brain barrier ; Horseradish peroxidase ; Senescence-accelerated mouse ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The ultrastructural features of microvessels showing increased permeability to intravenously injected horseradish peroxidase (HRP) were examined in the olfactory bulbs of senescence-accelerated prone mice (SAMP8), which showed age-related deficits in learning and memory, and senescence-accelerated resistant mice (SAMR1), which did not show the age-related deficits. HRP was visualized with tetramethyl benzidine (TMB) and diaminobenzidine (DAB) for light and electron microscopic examination, respectively. In the olfactory bulbs of 13-month-old SAMP8 mice, the staining reaction with TMB for HRP appeared in the neuropil of central area (granule cell layer and subependymal layer), in the pia mater and in the vascular wall. Some vessels located in the central area showed several changes observed at the ultrastructural level. The cytoplasm of the endothelial cells, especially in the arterioles, was segmentally thickened and contained numerous vesicles and vacuoles, some of which were HRP positive. The endothelial cell surface was occasionally undulated with microvillous protrusions. Membranous inclusions within the basal lamina, suggesting the cellular (presumably pericytal) degeneration, were frequently observed, especially in venules. The collagen deposits were occasionally observed in the subendothelial space of some vessels. Perivascular cells with vacuolated inclusions or lipid-like droplets were present around some vessels in the central area of the olfactory bulbs of aged SAMP8 mice. On the other hand, in the microvessels located in the areas negative for HRP-TMB reaction, except the vessel walls, the cytoplasm of the endothelial cells with smooth luminal surface was flattened and some vesicles located there contained HRP-DAB reaction product. Weak staining reaction with TMB for HRP appeared also in the central area of the olfactory bulbs of 3-month-old SAMP8 mice and 3- and 13-month-old SAMR1 mice. The cytoplasm of the endothelial cells in the olfactory bulbs of these mice was focally thickened and contained some cytoplasmic vesicles. Occasionally, the endothelial cell surface was moderately undulated with few microvillous protrusions. Membranous inclusions within the basal lamina were not observed in these animals. These findings indicate that the endothelial cells and pericytes in some vessels located in the central area of the olfactory bulb of aged SAMP8 mice, which show staining reaction with TMB for HRP, are ultrastructurally changed, suggesting their altered functions.
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  • 49
    ISSN: 1432-069X
    Keywords: Key words Barrier function ; pH ; Stratum corneum ; Lamellar body ; Lipid content ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Epidermal permeability barrier homeostasis requires the postsecretory processing of polar lipid precursors into nonpolar lipid products within the stratum corneum (SC) interstices by a family of lipid hydrolases. A specific requirement for β-glucocerebrosidase (β-GlcCer’ase), which exhibits a distinct acidic pH optimum, is particularly well documented. Therefore, we sought to determine whether the recovery of the barrier after acute insults requires acidification of the SC. We examined permeability barrier recovery by assessing changes in transepidermal water loss (TEWL), SC membrane ultrastructure utilizing ruthenium tetroxide (RuO 4 ) postfixation, and β-GlcCer’ase activity by in situ zymography at an acidic vs neutral pH. Barrier recovery proceeded normally when acetone-treated skin was exposed to solutions buffered to an acidic pH. In contrast, the initiation of barrier recovery was slowed when treated skin was exposed to neutral or alkaline pH, regardless of buffer composition. In addition, enhancement of the alkaline buffer-induced delay in barrier recovery occurred with Ca 2+ and K + inclusion in the buffer. Moreover, the pH-dependent alteration in barrier recovery appeared to occur through a mechanism that was independent of Ca 2+ - or K + -controlled lamellar body secretion, since both the formation and secretion of lamellar bodies proceeded comparably at pH 5.5 and pH 7.4. In contrast, exposure to pH 7.4 (but not pH 5.5) resulted in both the persistence of immature, extracellular lamellar membrane structures, and a marked decrease in the in situ activity of β-GlcCer’ase. These results suggest first that an acidic extracellular pH is necessary for the initiation of barrier recovery, and second that the delay in barrier recovery is a consequence of inhibition of postsecretory lipid processing.
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  • 50
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    Zeitschrift für Lebensmittel-Untersuchung und -Forschung 206 (1998), S. 38-43 
    ISSN: 1431-4630
    Keywords: Key words Starch ; Gels ; Kamaboko ; Surimi ; Gelatinization ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  The purpose of this work was to study the changes undergone by starch during heat-induced surimi gel preparation either with or without added egg white, and their effects on the structure of gels using light and scanning electron microscopy. Gels were made from SA-grade Alaska pollack (Theragra chalcogramma) surimi with: (1) salt (3%, w/w); (2) salt and waxy corn starch (3% and 5%, respectively w/w); or (3) salt, waxy corn starch and egg white (3%, 5% and 5%, respectively, w/w). Final moisture was adjusted to 73% or 83%. The gels were prepared by prior setting (40°C, 30 min, followed by 90°C, 30 min) or cooking (90°C, 30 min). The prepared gel was frozen and stored at –20°C (±1°C) until analysis. Samples were observed by light and scanning electron microscopy. The results show that the starch granules alter according to the processing conditions, with the predominance of crystalline or amorphous morphology depending upon the availability of heat and water. Large cavities formed in the protein gel matrix during setting can trap water; as a result, water availability is limited for starch to swell and gelatinize even in the high-moisture gel.
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  • 51
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    Der Pathologe 19 (1998), S. 373-378 
    ISSN: 1432-1963
    Keywords: Schlüsselwörter Metaplastische Brustdrüsenkarzinome ; Plattenepithelmetaplasie ; Plattenepithelkarzinom ; Immunhistologie ; Elektronenmikroskopie ; Zytophotometrie ; Key words Pure squamous cell carcinoma ; Mammary gland ; Squamous metaplasia ; Immunohistology ; Cytophotometry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Squamous metaplasia can be demonstrated in about 4% of all invasive carcinomas of the breast. Primary squamous cell carcinomas of the breast are rare, since they occur in less than 1% of all primary invasive breast carcinomas. In order to classify a breast tumor as a primary squamous cell carcinoma one must exclude an epidermal origin, especially from the nipple region and the possibility of metastatic infiltration of the breast by a squamous cell carcnoma from a different location. Causative and formal pathogenesis of primary squamous cell carcinoma of the breast is not clear. A pluripotent embryonal stem cell origin is discussed, considering the phylogenetic descent of the mammary gland from skin appendages. Squamous metaplasia is also suggested to be a precursor of squamous cell carcinoma. Here endocrine stimulation and chronic inflammation may both play an inductive role. The number of published cases of squamous cell carcinomas developing years and decades after implantation of silicon prostheses has increased in recent years. These tumors probably develop on top of squamous metaplasia induced by the inflammatory pseudocapsule. Estimating the prognosis and therapeutic management in patients with squamous cell carcinoma of the breast should follow the same guidelines as for other squamous cell cancers.
    Notes: Zusammenfassung Plattenepithelmetaplasien werden bei etwa 4% aller invasiven Brustdrüsenkarzinome beschrieben. Reine Plattenepithelkarzinome der weiblichen Brustdrüse sind mit einem Anteil von wahrscheinlich unter 1% an allen invasiven epithelialen Tumoren der Mamma selten. Von einem primären Plattenepithelkarzinom der Brustdrüse darf nur gesprochen werden, wenn zum einen der Ursprung von der Epidermis, insbesondere auch im Bereich des Mamillentrichters ausgeschlossen ist und zum anderen keine metastatische Infiltration in die Brustdrüse durch ein Plattenepithelkarzinom anderer Organlokalisation vorliegt. Die kausale und formale Pathogenese der primären Plattenepithelkarzinome der Brustdrüse ist unklar. Zum einen wird ein Ursprung von pluripotenten embryonalen Stammzellen diskutiert, wobei bedacht wird, daß die Brustdrüse entwicklungsgeschichtlich ein Hautanhangsgebilde darstellt. Zum anderen werden Plattenepithelmetaplasien als Vorstufe der Plattenepithelkarzinome diskutiert, wobei neben einer endokrinen Induktion auch länger bestehende Entzündungsreize eine Rolle spielen sollen. In den letzten Jahren wird immer häufiger darüber berichtet, daß oft Jahrzehnte nach Implantation von Silikonprothesen periprothetische Plattenepithelkarzinome entstehen, die wahrscheinlich über die Stufe einer Plattenepithelmetaplasie der entzündlichen Prothesenpseudokapsel entstehen. Die Abschätzung der Prognose und therapeutische Maßnahmen bei primären Plattenepithelkarzinomen der Brustdrüse sollten an den Erfahrungen mit Plattenepithelkarzinomen anderer Organlokalisation ausgerichtet werden.
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  • 52
    ISSN: 1432-2145
    Keywords: Key words Abies ; Egg cell ; Plastid inheritance ; RFLP ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The ultrastructure of egg cells in Abies alba was examined to elucidate the lack of maternal inheritance of plastids. Before fertilization, maternal plastids are absent in the perinuclar zone containing mainly mitochondria and smooth endoplasmic reticulum. During egg cell development the maternal plastids are transformed into large inclusions which are situated mostly towards the periphery of the egg cell, and finally disintegrate. As a consequence, they do not participate in zygote formation. RFLP analysis of cpDNA of parental trees and their F1 interspecific hybrids (A. alba×A. numidica, A. alba×A. nordmanniana, A. nordmanniana×A. Alba) using HindIII and BamHI showed a paternal mode of cpDNA inheritance. Paternal inheritance has also been found with PCR/RFLP analysis of cpDNA from parental trees and their hybrids (A. alba×A. pinsapo, A. pinsapo×A. alba, A. pinsapo×A. numidica) using ApaI and HaeIII digests, as well as in the crosses of A. cephalonica×A. nordmanniana, A. nordmanniana×A. cephalonica, A. cephalonica×A. numidica using TagI digests.
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  • 53
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    Sexual plant reproduction 11 (1998), S. 121-130 
    ISSN: 1432-2145
    Keywords: Key words Egg cell ; Parthenogenesis ; Synergid ; Ultrastructure ; Wheat ; Zygote
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line.
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  • 54
    ISSN: 1432-072X
    Keywords: Key words Magnetic bacteria ; Biomineralization ; Magnetite ; 16S rRNA ; In situ hybridization ; Ultrastructure ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.
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  • 55
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    Cell & tissue research 292 (1998), S. 275-282 
    ISSN: 1432-0878
    Keywords: Key words Interstitial cells (Cajal) ; Large intestine ; Fluorescent dyes ; Vital staining ; Ultrastructure ; Mouse (BALB/c)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 μm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3–6 main processes whose mean length was 56.3 μm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P〈0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues.
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  • 56
    ISSN: 1432-0878
    Keywords: Key words Cutaneous scent glands ; Apocrine glands ; Myoepithelial cells ; Holocrine glands ; Ultrastructure ; Lectins ; Cytokeratins ; Impala ; Aepyceros melampus (Artiodactyla)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The cutaneous glands of the forehead and the metatarsus were studied by histological and histochemical methods and electron microscopy in adult male and female impalas in various seasons of the year. All glandular areas consist of apocrine and holocrine glands, which, however, occur in different proportions. Our findings in the apocrine gland cells suggest (1) the synthesis and exocytosis of a glycoproteinaceous secretory product stored in secretory granules, (2) typical apocrine secretion of the transformed apical cytoplasm, and (3) transepithelial fluid transport. The Golgi apparatus and apical membrane have binding sites for several lectins (PNA, HPA, RCA I, WGA). Cytokeratins 7, 14 and 19 are expressed at various intracellular localizations, suggesting an active role in the secretory mechanisms. The glands of the male forehead show marked seasonal changes in activity that are correlated with the main phases of the reproductive cycle, with the highest cellular activity occurring during the rut in April/May. The female forehead glands are only moderately developed and do not undergo seasonal changes. The metatarsal glands are of equal size in males and females and show no seasonal changes in activity. This study supports the hypothesis that (1) forehead glands in the male have a signaling role in the rut and (2) the metatarsal glands have a more general, probably social role maintaining and restoring contact between herd members.
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  • 57
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    Cell & tissue research 293 (1998), S. 165-171 
    ISSN: 1432-0878
    Keywords: Key words Muscle injury ; Cytoskeleton ; Sarcomere organisation ; Immunohistochemistry ; Ultrastructure ; Rabbit (New Zealand White)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunohistochemical and electron-microscopic techniques were used to analyze the extensor digitorum longus muscles of New Zealand White rabbits 1 h, 1 day, 3, 7, and 28 days after repetitive eccentric contractions. Loss of the cytoskeletal protein desmin was the earliest manifestation of injury. Apart from 1 h post-exercise, all desmin-negative fibers stained positively with antibody to plasma fibronectin, indicating loss of cellular integrity accompanying cytoskeletal disruption. Fiber sizes were significantly increased from 1–7 days after exercise. The large (hyaline) fibers found in histological sections after repetitive eccentric contractions resulted from segmental hypercontraction of the fiber. This phenomenon occurred proximally and distally to plasma membrane lesions of the muscle fiber and necrosis and manifested itself as very short sarcomere lengths. Thus, in serial sections, staining characteristics, sizes and shapes of one and the same fiber often varied dramatically. We conclude that the following sequence of events occurs: cytoskeletal disruptions, loss of myofibrillar registry, i.e., Z-disk streaming and A-band disorganization, and loss of cell integrity as manifested by intracellular plasma fibronectin stain, hypercontracted regions, and invasion of cells. When a fiber is disrupted, the remaining intact fibers apparently take up the tension put on the muscle and later fewer fibers are subjected to eccentric contractions.
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    Cell & tissue research 294 (1998), S. 253-260 
    ISSN: 1432-0878
    Keywords: Key words P2X1 receptor ; Ultrastructure ; Cerebellum ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The distribution of the P2X1 subtype of purinoceptors associated with the extracellular activities of ATP was studied in the rat cerebellum at the electron-microscope level. Receptors were labelled with peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex for immunocytochemistry. Immunoreactivity to P2X1 receptors was localized in subpopulations of synapses between varicosities of parallel fibres of granule cells and dendritic spines of Purkinje cells. Unlabelled varicosities of parallel fibres formed asymmetric synapses with labelled dendritic spines, whereas labelled varicosities of parallel fibres formed asymmetric synapses with unlabelled dendritic spines. P2X1 immunoreactivity was also localized in some astrocyte processes. The functional significance of these findings is discussed.
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    Cell & tissue research 294 (1998), S. 367-375 
    ISSN: 1432-0878
    Keywords: Key words Neurons ; Glia ; Tracheae ; Wing ; Ultrastructure ; Moth ; Manduca sp.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  This ultrastructural examination of sensory nerves of the Manduca wing has revealed that extensive remodeling occurs among insect sensory neurons and their associated glial cells between pupation and adult emergence. Systematic counts of axons in particular wing nerves throughout adult development have shown that a decrease in axon number per nerve occurs after day 6. The neurons and glial cells that die are believed to be cells present at pupation that have no apparent sensory function but that probably function as guidance scaffolding for neurons and glia that are born after pupation. Despite the loss of several axons from each wing nerve, these nerves continue to grow in diameter during the latter half of adult development as some of the surviving axons increase severalfold in diameter. Each growing wing nerve in turn apparently functions as a scaffold for the proximal to distal growth of adult tracheae. A correspondence exists between adult nerve pathways and adult tracheal pathways, with each trachea maintaining intimate contact with a wing nerve along its entire length.
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  • 60
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    Cell & tissue research 291 (1998), S. 549-559 
    ISSN: 1432-0878
    Keywords: Key words Endolymphatic sac ; Ultrastructure ; Fluid transport ; Otoconia ; Newt ; Cynops pyrrhogaster (Urodela)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the “floccular” vesicle and the “granular” vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain flocccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.
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  • 61
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    Cell & tissue research 293 (1998), S. 253-269 
    ISSN: 1432-0878
    Keywords: Key words Septate junctions ; Ultrastructure ; Permeability ; Ions ; Epithelium ; Photoreceptor ; Hirudo medicinalis (Hirudinea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular “vacuole” that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial sections from photoreceptors infiltrated with colloidal lanthanum. The septa form tortuous interseptal corridors that are spatially continuous, and open ended basally and apically. Individual septa seem to be impermeable to lanthanum; interseptal corridors form the only diffusional pathway for this ion. The junctions form no diffusion barrier for the electron-dense tracer Ba2+, but they hinder the diffusion of various hydrophilic fluorescent dyes as demonstrated by confocal laser scanning microscopy (CLSM) of live cells. Even those dyes that penetrate gap junctions do not diffuse beyond the septate junctions. The aqueous diffusion pathway within the septal corridors is, therefore, less permeable than the gap-junctional pore. Our morphological results combined with published electrophysiological data suggest that the septa themselves are not completely tight for small physiologically relevant ions. We also examined, by CLSM, whether the septate junctions create a permeability barrier for the lateral diffusion of fluorescent lipophilic dyes incorporated into the peripheral membrane domain. AFC16, claimed to remain in the outer membrane leaflet, does not diffuse beyond the junctional region, whereas DiIC16, claimed to flip-flop, does. Thus, pleated septate junctions, like vertebrate tight junctions, contribute to the maintenance of cell polarity.
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  • 62
    ISSN: 1432-0878
    Keywords: Key words Angiogenesis ; Capillary growth ; Prazosin ; Shear stress ; Skeletal muscle ; Ultrastructure ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the α1-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: J v(c,f)=262±54 compared with 350±17 mm–2, control compared with prazosin (P〈0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5±0.9% after 2 weeks (P〈0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7±0.8% of controls; 7.1±1.9% at 1 week; 8.8±2.5% at 2 weeks; P〈0.02), some of which were smaller in size (less than 2 μm diameter) than in controls (3–5 μm) and/or “seamless”, i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5±6.2%, P〈0.001) and 2 weeks of prazosin (34.2±5.5%, P〈0.05) than in control vessels (16.7±3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks’ prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.
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  • 63
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    Protoplasma 204 (1998), S. 155-169 
    ISSN: 1615-6102
    Keywords: Spores ; Ultrastructure ; Entorrhiza ; Microbotryum ; Tilletia ; Ustilago
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The concept and nomenclature for the elements of teliospore walls in smut fungi are presented and a survey of teliosporogenesis is given, as seen by light and transmission electron microscopy. Four developmental types are distinguished: the Ustilago, Microbotryum, Tilletia, and Entorrhiza type. In the Ustilago type, sporogenous hyphae are completely segmented into teliospore initials which are embedded in a hyaline matrix formed by gelatinised hyphal walls (found in species ofAnthracoidea, Cintractia, Heterotolyposporium, Kuntzeomyces, Macalpinomyces, Melanopsichium, Sporisorium, Testicularia, Tolyposporium junci, Trichocintractia, and species ofUstilago infecting members of the family Poaceae). In the Microbotryum type, septate sporogenous hyphae are also completely segmented into teliospore initials, however, they are not surrounded by a hyaline matrix (Microbotryum, Sphacelotheca, Ustilago spp. infecting dicotyledons). A yeast-like budding of teliosporogenic cells is observed for some species ofMicrobotryum, Sphacelotheca, andUstilago infecting dicotyledons. In the Tilletia type, teliospores differentiate locally in the sporogenous hyphae, in an apical or intercalary position, without a hyaline matrix (Conidiosporomyces, Doassinga, Entyloma, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria, Rhamphospora, Tilletia). In all these types, the teliospore initials first develop a hyaline sheath under which the ornamentation, the exosporium, sometimes a middle layer, and the endosporium are successively deposited by the fungal cell. In the Entorrhiza type, the teliospores develop inside vital host cells with the wall of the sporogenous hypha included into the teliospore wall. The fungus develops a middle layer and an electron-transparent endosporium inside the hyphal wall while a layer forming the ornamentation is deposited onto the hyphal wall, probably by vesicles of dictyosomes of the host cell.
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  • 64
    ISSN: 1615-6102
    Keywords: Neck cell proliferation ; Binucleate ; Douglas fir ; Conifers ; Genetic instability ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary To study the effect of culturing on megagametophytes of Douglas fir (Pseudotsuga menziesii) and hybrid larch (Larix × eurolepis), cones were collected at the time of fertilization and the megagametophytes were removed, then placed on medium. We used a modified Murashige and Skoog medium supplemented with 5% lactose and 10% polyethylene glycol 4000. A variety of cell types proliferated including prothallial, neck, and jacket cells. Some of these multiplying cells showed a binucleate condition. The prothallial cells of the apex divided and expanded. The neck cells formed clusters composed of more cells than normally found in situ; though otherwise they showed ultrastructural similarity to neck cells in situ. These neck cells had large numbers of active Golgi complexes, numerous large and small vacuoles, coated vesicles, smooth vesicles, a well-developed endoplasmic reticulum, and thickened cell walls. These are the first reports of neck cell multiplication and induction of a binucleate state for gymnosperm megagametophyte cells in vitro.
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  • 65
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    Protoplasma 201 (1998), S. 30-37 
    ISSN: 1615-6102
    Keywords: Cell-to-cell communication ; Plasmodesmata ; Ultrastructure ; Wounding ; 2-Deoxy-D-glucose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The transport of ions and metabolites through plasmodesmata has been thought to be controlled at the neck region where the cytoplasmic annulus is constricted and where callose has also been localised. In order to determine the possible structural and functional effects of callose, its deposition was inhibited through incubation of the plant tissue with 2-deoxy-D-glucose (DDG) for 1 h prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the plasmodesmata fromAllium cepa L. roots treated with DDG exhibited a funnel-shaped configuration. This is in contrast to the plasmodesmata from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported. Both initial dissection and glutaraldehyde fixation induced neck constriction in plasmodesmata, however, dissection of tissue increased the frequency of constrictions. The inhibition of callose formation by chemical means showed that the neck constrictions and raised collars in this area are artefacts due to physical wounding and glutaraldehyde fixation. The external electron-dense material observed when tannic acid is included in the primary fixative appears to be unrelated to the deposition of callose at the neck region.
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  • 66
    ISSN: 1615-6102
    Keywords: Chloroplast development ; Cotton ; Fluorescence induction kinetics ; Ultrastructure ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Many of the studies of chloroplast ontogeny in higher plants have utilized suboptimal conditions of light and growth to assess development. In this study, we utilized structural, immunological, and physiological techniques to examine the development of the chloroplast in fieldgrown cotton (Gossypium hirsutum cv. “MD 51 ne”). Our youngest leaf sample developmentally was completely folded upon itself and about 0.5 cm in length; leaves of this same plastochron were followed for three weeks to the fully expanded leaf. The chloroplasts at the earliest stage monitored had almost all of the lamellae in small, relatively electron-opaque grana, with relatively few thylakoids which were not appressed on at least one surface. During the development of the thylakoids, the membranes increase in complexity, with considerable stroma lamellae development and an increase in the number of thylakoids per granum. Besides the increase in complexity, both the size and numbers of the chloroplast increase during the development of the leaf. Developmental changes in six thylakoid proteins, five stromal proteins, and one peroxisomal protein were monitored by quantitative immunocytochemistry. Even at the earliest stages of development, the plastids are equipped with the proteins required to carry out both light and dark reactions of photosynthesis. Several of the proteins follow three phases of accumulation: a relatively high density at early stages, a linear increase to keep step with chloroplast growth, and a final accumulation in the mature chloroplast. Photosystem-II(PS II)-related proteins are present at their highest densities early in development, with an accumulation of other parts of the photosynthetic apparatus at a latter stage. The early accumulation of PS-II-related proteins correlates with the much lower ratio of chlorophylla tob in the younger leaves and with the changes in fluorescence transients. These data indicate that some of the conclusions on chloroplast development based upon studies of intercalary meristems of monocots or the greening of etiolated plants may not be adequate to explain development of chloroplasts in leaves from apical meristems grown under natural conditions.
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  • 67
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    Protoplasma 201 (1998), S. 101-109 
    ISSN: 1615-6102
    Keywords: Kinetochore ; Kinetoplastida ; Intranuclear microtubules ; Mitosis ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The three-dimensional ultrastructural organization of the mitotic apparatus ofDimastigella mimosa was studied by computer-aided, serial-section reconstruction. The nuclear envelope remains intact during nuclear division. During mitosis, chromosomes do not condense, whereas intranuclear microtubules are found in close association with six pairs of kinetochores. No discrete microtubule-organizing centers, except kinetochore pairs, could be found within the nucleus. The intranuclear microtubules form six separate bundles oriented at different angles to each other. Each bundle contains up to 8 tightly packed microtubules which push the daughter kinetochores apart. At late anaphase only, midzones of these bundles align along an extended interzonal spindle within the narrow isthmus between segregating progeny nuclei. The nuclear division inD. mimosa can be described as closed intranuclear mitosis with acentric and separate microtubular bundles and weakly condensed chromosomes.
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  • 68
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    Plant systematics and evolution 210 (1998), S. 25-39 
    ISSN: 1615-6110
    Keywords: Cyanophyta ; Cyanobacteria ; Cyanothece ; Synechococcus ; Cyanobium ; Ultrastructure ; nucleoids ; taxonomy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The fine structure of the type species of the genusCyanothece Komárek 1976,C. aeruginosa, is described and compared with the main cytological characteristics of morphologically related members of the generaCyanobium, Cyanobacterium andSynechococcus. Several morphological features, such as cell walls with thick outer layers containing a special type of vesicles, position of thylakoids, “keritomy” (net-like appearance of protoplast caused by arrangement of thylakoids, net-like nucleoids and/or by tendency to form intrathylakoidal spaces) and a special structure of mucilaginous envelopes were found to be characteristic of this genus, supporting its separate position among coccal cyanoprokaryotes (cyanobacteria, cyanophytes). The taxonomic significance of ultrastructural features in all mentioned genera is discussed.
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  • 69
    ISSN: 1615-6102
    Keywords: Spore balls ; Germ areas ; Ultrastructure ; Phylogeny
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Special features of teliospores in smut fungi are described, including teliospore connections, appendages, and germ pores. Balls of teliospores in species of many different genera cohere by remnants of hyphal walls, sheaths, and sometimes interlocking ornamentation. Teliospores are connected in pairs in species ofMycosyrinx andGeminago by special local structures. Appendages can be formed locally by persistent material from the sheath (Cintractia, Anthracoidea, Sphacelotheca), thickened parts of the spore wall (e.g.,Georgefischeria, Jamesdicksonia, Rhamphospora, Tolyposporella), or persistent walls of sporogenous hyphae (Rhamphospora, genera of the Tilletia relationship). Species ofGeorgefischeria, Jamesdicksonia, andTolyposporella have teliospore walls composed of more than three layers of different electron density. “Germ areas” corresponding to thinner parts of the spore wall are known, e.g., for species ofAnthracoidea, Cintractia, andUstilago infecting members of the family Poaceae, while distinct germ pores, one per teliospore, are found in some species ofThecaphora, “Tolyposporium”, andSporisorium. Teliospores ofMycosyrinx cissi have a germination ring. Characteristics of teliospores are used to discuss the phylogeny of smut fungi. A phylogenetic tree in accordance with teliospore characteristics is compared to those obtained from ultrastructural characteristics of host-parasite interaction, of septal pores, and from sequence data. Aspects of teliospore development help to define taxa at a high systematic level (Entorrhizales, Ustilaginales, Tilletiales/Entylomatales, Microbotryaceae), while details of ornamentation ontogeny delimit groups of genera (e.g., genera related toUstilago on members of the Poaceae andSporisorium, Cintractia andAnthracoidea, Tilletia) or single genera (e.g.,Melanopsichium, Dermatosorus, Mycosyrinx, Doassinga, Rhamphospora). Types of ornamentation (warty, reticulate), middle layers, teliospore balls, and germ pores evolved repeatedly by convergence. The smut teliospore itself probably evolved independently at least twice, or perhaps three (or more) times, in the Microbotryales, in the Entorrhizales, and in a common ancestor of the remainder of the Ustilaginomycetes.
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  • 70
    ISSN: 1615-6102
    Keywords: Spores ; Ultrastructure ; Microbotryum ; Tilletia ; Tolyposporium ; Ustilago
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The walls of mature teliospores and the development of ornamentation, as seen by transmission electron microscopy, are described for 37 genera of smut fungi, based on observations of ca. 120 species and on literature. Structural diversity of mature teliospore walls is due to differences in spore wall layers forming the spore wall (endosporium, middle layer, exosporium, ornamentation) and to different elements forming the ornamentation (exosporium, ornaments, sheath, hyphal wall, adjacent fungal cells, material of the host). During teliosporogenesis the outer layers are usually deposited first. At the beginning of the formation of the ornamentation the plasma membrane may be smooth or undulated carrying the developing ornaments on its tips or in its depressions. The ornamentation of some genera appears similar when seen by scanning electron microscopy, but can be the product of different developmental patterns (e.g., warts of species ofFarysia, Tilletia, andUstilago), however, warty and reticulate ornamentation can both be produced by similar developmental processes (shown, e.g., for species ofCintractia andTilletia). Typical structures of the mature teliospore wall and developmental patterns based on homologous similarities are described for the following groups of genera or species:Macalpinomyces, Melanopsichium, Sporisorium, andUstilago infecting members of the family Poaceae;Kuntzeomyces, Testicularia, andTrichocintractia; Anthracoidea, Cintractia, Heterotolyposporium piluliforme, andTolyposporium junci; Glomosporium, Sorosporium, andThecaphora; Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria, andTilletia; Entyloma, and genera of the Doassansia group;Liroa, Microbotryum, Sphacelotheca, Ustilago infecting dicotyledons, andZundeliomyces; Aurantiosporium, Fulvisporium, andUstilentyloma. Special characteristics of the teliospore wall were observed for the generaDermatosorus, Doassinga, Entorrhha, Farysia, Mycosyrinx, Rhamphospora, and some species ofTolyposporium.
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    Biotechnology and Bioengineering 57 (1998), S. 590-599 
    ISSN: 0006-3592
    Keywords: protein refolding ; hollow-fibre membrane ; dialysis ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25°C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25°C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 590-599, 1998.
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  • 72
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    Biotechnology and Bioengineering 58 (1998), S. 119-120 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 73
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    Biotechnology and Bioengineering 58 (1998), S. 658-662 
    ISSN: 0006-3592
    Keywords: T4 lysozyme ; silica nanoparticles ; synthetic enzyme variants ; surface-induced conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Maintaining a specific molecular conformation is essential for the proper functioning of an enzyme. A substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution. Activity may also be lost as an enzyme undergoes a conformational change during adsorption. In this study, we investigated the effect of thermostability on the activities of three T4 lysozyme variants after adsorption to 9 nm colloidal silica particles. Less-stable T4 lysozyme variants lost more activity after adsorption than did more stable variants, apparently because they experienced more extensive structural alteration. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 658-662, 1998.
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  • 74
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    Biotechnology and Bioengineering 58 (1998), S. 139-148 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; pathway analysis ; metabolic and energetic model ; physiological state ; Saccharomyces cerevisiae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose. This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth. The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture. Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy. The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity. The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop. All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:139-148, 1998.
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  • 75
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    Biotechnology and Bioengineering 58 (1998), S. 149-153 
    ISSN: 0006-3592
    Keywords: Metabolic Control Analysis ; flux control coefficients ; top down MCA ; metabolic engineering ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group (flux, as well as concentration) control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, we demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmental perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:149-153, 1998.
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  • 76
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    Biotechnology and Bioengineering 58 (1998), S. 154-161 
    ISSN: 0006-3592
    Keywords: central carbon pathways ; metabolic optimization ; ethanol production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:154-161, 1998.
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  • 77
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    Biotechnology and Bioengineering 58 (1998), S. 170-174 
    ISSN: 0006-3592
    Keywords: catabolite repression ; phosphotransferase system ; inducer exclusion ; inducer expulsion ; protein kinase ; transcriptional regulation ; transport regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catabolite repression is a universal phenomenon, found in virtually all living organisms. These organisms range from the simplest bacteria to higher fungi, plants, and animals. A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms. However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives. Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E. coli. In fact, single-celled organisms such as E. coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent. The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:170-174, 1998.
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  • 78
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    Biotechnology and Bioengineering 58 (1998), S. 162-169 
    ISSN: 0006-3592
    Keywords: bioinformatics ; metabolic engineering ; genetic engineering ; mathematical analysis ; stoichiometry ; enzyme kinetics ; modal analysis ; genetic circuits ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:162-169, 1998.
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  • 79
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    Biotechnology and Bioengineering 58 (1998), S. 191-195 
    ISSN: 0006-3592
    Keywords: control analysis ; Lactococcus lactis ; gene expression ; flux ; oligonucleotide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level. One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:191-195, 1998.
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  • 80
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    Biotechnology and Bioengineering 58 (1998), S. 175-190 
    ISSN: 0006-3592
    Keywords: protein-based polymers ; inverse temperature transitions ; hydrophobic-induced pKa shifts ; waters of hydrophobic hydration ; five axioms for protein engineering; microwave dielectric relaxation ; a universal mechanism for biological energy conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill.This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions.Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity.Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:175-190, 1998.
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  • 81
    ISSN: 0006-3592
    Keywords: Escherichia coli ; Chloramphenicol Acetyltransferase (CAT) ; Culture Redox Potential (CRP) ; Dithiothreitol (DTT) ; reducing agents ; molecular chaperones ; proteases ; heat shock ; stress response ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248-259, 1998.
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  • 82
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    Biotechnology and Bioengineering 59 (1998), S. 261-272 
    ISSN: 0006-3592
    Keywords: effective diffusive permeability ; diffusion coefficient ; biofilm ; cell density ; review ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes ( 〉 5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:261-272, 1998.
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  • 83
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    Biotechnology and Bioengineering 59 (1998), S. 281-285 
    ISSN: 0006-3592
    Keywords: protein aggregation ; RNase A ; protein formulation ; protein additives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the protein did not aggregate even when exposed to 75°C for 24 h. In this study, electrostatic repulsion was shown to be responsible for the absence of aggregates at that pH. While RNase A aggregation was prevented at the extremely acidic pH, this is not an environment conducive to maintaining protein function in general. Therefore, attempts were made to confer electrostatic repulsion near neutral pH. In this study, heat-denatured RNase A was mixed with charged polymers at pH 7.8 in an attempt to provide the protein with excess surface cations or anions. At 75°C, SDS and dextran sulfate were successful in preventing RNase A aggregation, whereas their cationic, nonionic, and zwitterionic analogs did not do so. We believe that the SO3- groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion at pH 7.8 and 75°C to prevent aggregation from proceeding. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:281-285, 1998.
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  • 84
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    Biotechnology and Bioengineering 59 (1998), S. 328-343 
    ISSN: 0006-3592
    Keywords: biotrickling filters ; biotrickling filter modeling ; mono-chlorobenzene ; biodegradation kinetics of mono-chlorobenzene ; chlorinated VOC emissions ; biofiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of mono-chlorobenzene (m-CB) vapor from airstreams was studied in a biotrickling filter (BTF) operating under counter-current flow of the air and liquid streams. Experiments were performed under various values of inlet m-CB concentration, air and/or liquid volumetric flow rates, and pH of the recirculating liquid. Conversion of m-CB was never below 70% and at low concentrations exceeded 90%. A maximum removal rate of about 60 gm-3-reactor h-1 was observed. Conversion of m-CB was found to increase as the values of liquid and air flow rate increase and decrease, respectively. The effects of pH and frequency of medium replenishment on BTF performance were also investigated. The process was successfully described with a detailed mathematical model, which accounts for mass transfer and kinetic effects based on m-CB and oxygen availability. Solution of the model equations yielded m-CB and oxygen concentration profiles in all three phases (airstream, liquid, biofilm). It is predicted that oxygen has a controling effect on the process at high inlet m-CB concentrations. From independent, suspended culture, experiments it was found that m-CB biodegradation follows Andrews inhibitory kinetics. The kinetic constants were found to remain practically unchanged after the culture was used in BTF experiments for 8 months. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:328-343, 1998.
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  • 85
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    Biotechnology and Bioengineering 59 (1998), S. 344-350 
    ISSN: 0006-3592
    Keywords: electrodialysis ; citric acid ; pH ; temperature ; Faraday efficiency ; solute recovery efficiency ; specific energy consumption ; solute flux ; water flux ; feed solute concentration ; electric current density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of pH and temperature (θ) on the overall performance indicators (i.e., solute recovery, ρ, and Faraday, η, efficiencies; specific energy consumption, ε, solute, JS, and water, JW, fluxes) of batch electrodialytic recovery of citric acid from model solutions was assessed at different values of feed solute concentration (cSf) and electric current density (j). Regardless of the initial feed concentration used, ρ and JS were found to be independent of θ; η and JW exhibited a positive trend with respect to θ, while ε a negative one. At the maximum temperature tested (33°C), as the pH of the feed solution was varied from 3 to 7, ρ increased from 0.90 ± 0.08 to 0.97 ± 0.02, η grew from 0.09 ± 0.02 to 0.50 ± 0.01, JS practically doubled, ε reduced about 8 times, but JW increased from 3 to 4 times. So, the optimal conditions for this technique are to be determined by balancing the savings in the investment and maintenance costs against the energy costs. © John Wiley & Sons, Inc. Biotechnol Bioeng 59:344-350, 1998.
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  • 86
    ISSN: 0006-3592
    Keywords: chymotrypsin ; enzyme stability ; reversed micelles ; interface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of α-chymotrypsin and δ-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. α-Chymotrypsin is inactivated at the interface and at the water pool, while δ-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in δ-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since δ-chymotrypsin does not have a bell-shaped curve as observed for α-chymotrypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:360-363, 1998.
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  • 87
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    Biotechnology and Bioengineering 59 (1998), S. 351-359 
    ISSN: 0006-3592
    Keywords: bioreactor ; high density ; insect cells ; perfusion ; Sf9 ; ultrasonic filter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:351-359, 1998.
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  • 88
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    Biotechnology and Bioengineering 59 (1998), S. 374-378 
    ISSN: 0006-3592
    Keywords: conductive paint electrode ; prevention of marine biofouling ; fishing net ; alternating potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conductive paint electrode was used for marine biofouling on fishing nets by electrochemical disinfection. When a potential of 1.2 V vs. a saturated calomel electrode (SCE) was applied to the conductive paint electrode, Vibrio alginolyticus cells attached on the electrode were completely killed. By applying a negative potential, the attached cells were removed from the surface of the electrode. Changes in pH and chlorine concentration were not observed at potentials in the range -0.6 ∼1.2 V vs. SCE. In a field experiment, accumulation of the bacterial cells and formation of biofilms on the electrode were prevented by application of an alternating potential, and 94% of attachment of the biofouling organisms was inhibited electrically on yarn used for fishing net coated with conductive paint. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:374-378, 1998.
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  • 89
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    Biotechnology and Bioengineering 59 (1998), S. 364-373 
    ISSN: 0006-3592
    Keywords: porous supports ; internal and external diffusion ; active site accessibility ; enzyme loading ; kinetically controlled dipeptide synthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mass transfer limitations were studied in enzyme preparations of α-chymotrypsin made by deposition on different porous support materials such as controlled pore glasses, Celite, and polyamides of different particle sizes. It is the onset of mass transfer limitations that determines the position of the activity optimum with respect to enzyme loading on each support. The evidence of various experiments indicates that internal diffusional limitations are the important mechanism for the observed mass transfer limitations. External diffusion was not found to play an important role under the conditions used, and it was also found that when immobilizing multilayers of enzyme the buried enzyme molecules are active to a large extent. An extreme situation is observed on Celite at very high loadings. Under these conditions, this support is expected to have its pores completely filled with packed enzyme molecules, and then it is the diffusion within the enzyme layer that determines the observed rate. As the enzyme loading increases, the area of contact between the deposited enzyme layers and the liquid solution inside the pores diminishes, causing a decrease on the observed rate of an intrinsically fast reaction which apparently is incongruous with the presence of more enzyme in the system. This work shows that mass transfer limitations can be an important factor when working with immobilized enzymes in organic media, and its study should be carried out in order to avoid undesired reduced enzyme activities and specificities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:364-373, 1998.
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  • 90
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    Biotechnology and Bioengineering 59 (1998), S. 438-444 
    ISSN: 0006-3592
    Keywords: bioremediation ; plasma discharge ; dichlorophenol degradation ; perchloroethylene degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pulsed electric discharge (PED) and bioremediation were combined to create a novel two-stage system which dechlorinates the halogenated pollutants, 2,4-dichlorophenol and perchloroethylene, with repetitive (0.1-1 kHz), short pulse (∼100 ns), low voltage (40-80 kV) discharges and then mineralizes the less chlorinated products with aerobic bacteria. A 6.1 mM aqueous dichlorophenol sample was cycled through the PED reactor (60 kV of applied pulsed voltage and 300 Hz) 6 times, resulting in the release of 55% of the initial dichlorophenol chloride ions (1 mM Cl- removed each cycle). The respective average specific efficiency is 0.4-0.6 keV/(Cl- molecule). Pseudomonas mendocina KR1, which grows in minimal medium supplemented with phenol but not with dichlorophenol, increased in cell density in all cultures supplemented with the PED-treated DCP samples and yielded a maximum of two-fold additional Cl- released compared to the PED-related alone. The number of PED-treatment cycles, voltage, and frequency were also varied, showing that both cell densities and overall dichlorophenol dechlorination were highly dependent upon the number of PED-treatment cycles, rather than the tested voltages and frequencies. Using this two-stage treatment system, PED released 31% of the initial chloride ions from dichlorophenol (after three cycles at 40-45 kV and 1.2 kHz) while P. mendocina KR1 in the second stage increased dechlorination to 90%. These results were corroborated by the 35% additional chloride release found with activated sludge cultures. Perchloroethylene (0.6 mM) was similarly treated in a first-stage PED reactor (80% chloride removal after four cycles) followed by biodegradation of the dechlorinated products with a recombinant toluene o-monooxygenase-expressing Pseudomonas fluorescens strain. Gas chromatographic analysis showed that the PED reactor created less-chlorinated byproducts (i.e., trichloroethylene) that were removed (74%) upon exposure to the recombinant bacterium. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:438-444, 1998.
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  • 91
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    Biotechnology and Bioengineering 59 (1998), S. 445-450 
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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  • 92
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    Biotechnology and Bioengineering 59 (1998), S. 451-460 
    ISSN: 0006-3592
    Keywords: protein fouling ; membrane transport ; ultrafiltration ; adsorption ; filtration ; composite membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:451-460, 1998.
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  • 93
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    Biotechnology and Bioengineering 59 (1998), S. 461-470 
    ISSN: 0006-3592
    Keywords: aqueous two-phase separation ; protein partitioning ; T4 lysozyme ; electrochemical partitioning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components.We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:461-470, 1998.
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  • 94
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    Biotechnology and Bioengineering 57 (1998), S. 518-528 
    ISSN: 0006-3592
    Keywords: ammonium ; UDP-GlcNAc ; N -glycosylation ; BHK-21 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal α1-6-linked fucose; 37% of the antenna were found to be substituted with terminal α2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 518-528, 1998.
    Additional Material: 5 Ill.
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  • 95
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 557-570 
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; polyhydroxyalkanoates ; metabolic engineering ; mathematical modeling ; enzyme kinetics ; regulation of metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557-570, 1998.
    Additional Material: 7 Ill.
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  • 96
    ISSN: 0006-3592
    Keywords: rhG-CSF ; fusion protein ; secretion efficiency ; glycosylation ; multimer ; conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 600-609, 1998.
    Additional Material: 8 Ill.
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  • 97
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 620-623 
    ISSN: 0006-3592
    Keywords: protein refolding ; reversed micelles ; solid-liquid extraction ; RNase A ; DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 620-623, 1998.
    Additional Material: 3 Ill.
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  • 98
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 610-619 
    ISSN: 0006-3592
    Keywords: dynamic model ; Saccharomyces cerevisiae ; oxidative capacity ; feedback control ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism. Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism. Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit. The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value. It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant τ. The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions. On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism. The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized. Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters. Therefore, a proportional feedback controller using heat evolution rate measurements was implemented. Calorimetry provides an excellent and rapid estimate of the metabolic activity. Satisfactory control was achieved and led to constant biomass yields. Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 610-619, 1998.
    Additional Material: 9 Ill.
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  • 99
    ISSN: 0006-3592
    Keywords: c-jun ; cell cycle ; apoptosis ; antisense ; growth deprivation ; F-MEL ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 106 cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.© 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:65-72, 1998.
    Additional Material: 6 Ill.
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  • 100
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 380-386 
    ISSN: 0006-3592
    Keywords: reverse micelles ; cutinase ; deactivation ; conformational changes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380-386, 1998.
    Additional Material: 2 Ill.
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