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  • 2020-2022
  • 1990-1994  (878)
  • 1975-1979  (299)
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  • 1800-1809
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  • 1
    ISSN: 1432-2307
    Keywords: Human cutaneous leishmaniasis ; Skin ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary To characterize the in situ cellular immune response in localized cutaneous leishmaniasis (LCL), the authors studied frozen skin biopsies from 50 patients with LCL due toLeishmania braziliensis guyanensis. A panel of 31 monoclonal antibodies was used, which defined the number and distribution of inflammatory cell subsets. Skin inflammatory infiltrates were composed of T cells (with a local CD4/CD8 ratio of 1.05±0.7 vs 1.48±0.3 in peripheral blood), macrophages and a smaller number of B cells, natural killer cells and granulocytes. Most of the T cells expressed activation markers (interleukin-2 and transferrin receptors, HLA-DR+) and an increase in T-cell-receptorγδ expression was noted. Analysis of the CD4+ subpopulations with newly available reagents showed that helper T cells (CD4+CD45RO+) exceeded the suppressor/inducer subset (CD4+CD45RA+) by 1.4∶1. There were no differences between local immune variables from patients with primary infection (45 patients) and those with recurrence (5). In 7 patients, biopsies were analysed before and 1 month after specific treatment, and did not show significant differences except for a small increase of dermal CD1a+ (Langerhans) cells/mm2. The observed pattern of cellular skin infiltration suggests an immune-mediated tissue injury including T-cell-mediated cytotoxicity and delayed hypersensitivity reactions in addition to direct parasitic action.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1440
    Keywords: Kidney ; Glomerulus ; Mesangium ; Mesangial failure ; Electron microscopy ; Animal models
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We present a structural analysis of the ability of the biomechanical unit consisting of mesangium and glomerular basement membrane to maintain normal capillary architecture in the face of mechanical challenges due to high intraglomerular pressures. Capillary support function may be considered in terms of the stabilization of local form (development of wall tension against capillary dilation) and global form (centripetal fixation of capillary loops to maintain higher order form). The pathologic consequences of the loss of this support are illustrated by way of experimental models of mechanical mesangial failure. Such failure may express itself as mesangial widening, increased transmesangial macromolecule “traffic,” ballooning of capillary segments, and unfolding of capillary loops. Mechanisms are described by which these structural changes may lead to segmental glomerular sclerosis.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    International journal of legal medicine 105 (1992), S. 173-175 
    ISSN: 1437-1596
    Keywords: Human brain tissue ; Natural mummification ; Histology ; Histochemistry ; Electron microscopy ; Elemetal analysis ; Gehirn ; Mensch ; Naturaliche Mumifikation ; Histologie ; Histochemie ; Elektronenmikroskopie ; Atmospektralanalyse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Law
    Description / Table of Contents: Zusammenfassung Bei Ausgrabungen in Bulgarien wurden Schädel und Knochen von 39 Individuen im Alter zwischen 35 und 60 Jahren, die vor ca. 45–50 Jahren begraben wurden, gefunden. Bei der nachfolgenden gerichtsmedizinischen Experitse wurden in zwei intakten Schädeln und zwischen den Knochen anderer Schädel sieben harte Gebilde (zwei ganze und fünf Fragmente), die wie geschrumpfte menschliche Gehirne aussahen, identifiziert. Entsprechende Proben von diesem Material wurden für 3–4 Tage mit 15% wässriger Lösung von Glycerol weicher gemacht und danach lichtmikroskopisch und elektronenmikroskopisch untersucht. An entsprechenden Stückchen von frischem menschlichem Gehirn und den gefundenen Proben wurde eine Atomspektral-analyse zur Bestimmung des chemischen Bestandes an Elementen durchgeführt. Die komplexe Untersuchung des Materials ergab, daß es sich bei diesem Fund um einen Sonderfall von natürlich mumifizierten menschlichen Gehirnen handelt, die durch sonderbare Bedingungen nur im Schädel der begrabenen Individuen entstanden sind.
    Notes: Summary When skulls and bones were exhumed from a mass grave in Bulgaria and subjected to medicolegal examination they were found to originate from 39 humans aged 36–60 years old who had been buried approximately 45–50 years ago. Solid structures which strongly resembled shrunken human brain tissue were found inside 2 intact skulls. Among other bones 5 similar structures were found one of which was an almost entirely preserved human brain, and the others were fragments from different regions of the human brain. Samples of these structures were immersed in 15% aqueous glycerol solution to soften and were examined by light and electron microscopy. Sampels of this material and of fresh human brain were subjected to elementary atomic spectral analysis. These complex studies indicated the samples to be naturally mummified human brain tissue and that this process had occurred due to specific conditions within the cranial cavities after burial.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-2307
    Keywords: Carnitine-palmityl-transferase deficiency ; Acute vascular lesions ; Acute myofibrillar lesions ; Ischaemia ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A case of severe exercise-induced myoglobinuria in a 14-year-old boy suffering from a carnitine-palmityl-transferase (CPT) defect is reported. Biopsies of the forearm muscle were examined using light and electron microscopy in the acute and recovery phases of the illness. The first biopsy showed the presence of scattered foci of necrosis where necrotic fibres with occasional disruptions of the basal lamina were seen around injured capillaries. Various degrees of damage and different stages of evolution were found in these foci, which also contained regenerating muscle fibres. In the second biopsy, performed 2 weeks later, most of the fibres displayed a normal structure. Necrosis was no longer present. However, in some areas perivascular fibrosis was prominent, the fibres were small and irregularly shaped, and their nuclei often centrally located. These data strongly suggest that circulatory disorders and ischaemia, brought about by premature acute metabolic imbalance, could be involved in the development of exerciseinduced myolysis observed in CPT deficiency. The risk of fibrous cardiomyopathy in these patients is pointed out.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Virchows Archiv 421 (1992), S. 105-113 
    ISSN: 1432-2307
    Keywords: Parotid gland ; ras ; Transgenic mouse ; Electron microscopy ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In the transgenic TG.SH (mouse mammary tumour virus/v-Ha-ras) mouse, designed to develop mammary tumours, occasional spontaneous salivary gland tumours have been reported, predominantly in males. The incidence and histomorphology of salivary gland tumours in 73 TG.SH mice were surveyed and in total, 21.9% developed both overt and microscopic parotid tumours. The majority developed between 73 and 150 days of age. In 31.5% of the TG.SH mice, occasional unilateral, but more frequently bilateral exophthalmos due to hyperplasia of the intraorbital (Harderian) lacrimal gland was observed. In 70% of these animals, parotid tumours developed later. Since Harderian gland hyperplasia, occurring as early as 5 weeks of age, preceded the development of palpable salivary gland lesions, this stigma is useful for the early selection of animals likely to progress to tumour formation. Before tumour-bearing transgenic mice are considered to be suitable models of human neoplastic disease, morphological characterization is necessary to ensure that the tumours are histologically representative of the human lesions for which they are potential models. In this study, all parotid tumours consisted of acinar-like glandular structures with central lumina discernible by electron microscopy. Ultrastructurally, secretory granules evident in the apical cytoplasm of the tumour cells resembled the zymogen granules of the normal parotid acinar cell, and some cells had a prominent complement of rough endoplasmic reticulum. These features, along with focal amylase expression detected immunohistochemically in some parotid tumours, identified these neoplasms as acinic cell carcinomas that mimic the human salivary gland acinic cell carcinoma faithfully.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-2307
    Keywords: Silver staining ; Paired helical filaments ; Cerebral amyloid ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A one step en bloc silver staining method which was originally established to study nucleolar organizer regions has been applied for the demonstration of both paired helical filaments (PHF) and extracellular cerebral amyloids in semi-thin sections and at the electron microscopic level. The three forms of PHF can be visualized: (1) neurofibrillary tangles are shown in all stages from first appearance in form of intracellular patches of PHF to severely degenerated shadow-like “ghost” tangles; (2) neuropil threads are distinctly stained in great numbers; and (3) PHF are easily detected as neuritic components in amyloid plaques. All forms of fibrillar extracellular amyloid structures, i.e. “diffuse”, “classical” and “burnt out” plaques, are well demonstrated; congophilic angiopathy reveals amyloid preferentially in arteries and arterioles of the leptomeninges and cortex ranging from small circumscribed patches to large circumferential amounts with occasional plaque-like condensations or broad loose accumulations of amyloid; perivascular cuffs and laminar subpial deposits of amyloid are stained as well. At the electron microscopic level all lesions are clearly visible in non uranyl/lead-stained specimens, characterized by varying numbers of silver grains on a pale background. The detailed demonstration of structures in archival material, which had been stored in paraffin and re-embedded for electron microscopy, is due to the demonstration of argyrophilic structures by the protective colloidal developer of gelatin and formic acid and to the proteolytic resistance of insoluble PHF and extracellular amyloids in plaques and congophilic angiopathy.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Virchows Archiv 420 (1992), S. 453-457 
    ISSN: 1432-2307
    Keywords: Uterus ; Paraganglioma ; Intracytoplasmic hyaline globules ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We report a malignant uterine paraganglioma in a 40-year-old female, who died 7 months after the initial diagnosis. On light microscopy the tumour showed a typicalzellballen pattern as well as a pronounced cellular pleomorphism. In many tumour cells hyaline globules were demonstrated within the cytoplasm. Immunohistochemically the lesion was characterized by the presence of neuron-specific enolase, protein gene product 9.5 and synaptophysin, and electron microscopically by the occurrence of neurosecretory granules.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-0533
    Keywords: Peripheral neuropathy ; Cryoglobulinemia ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Histological, immunohistochemical and ultrastructural sural nerve and skin biopsy findings in a case of cryoglobulinemia secondary to an IgM-kappa-producing non-Hodgkin lymphoma are described. The main finding was an occlusive microangiopathy present in both the sural nerve and the skin. Widespread cryoglobulin deposits of the proliferated vasa nervorum were associated with pronounced changes probably evoked by ischemia. Moderate perivascular inflammation, but no florid vasculitis was additionally present. Our observations indicate that occlusive microangiopathy by precipitated cryoglobulins may be a relevant pathogenetic factor in cryoglobulinemic peripheral neuropathy.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-0533
    Keywords: Progressive supranuclear palsy ; Neurofibrillary tangles ; Pick body ; Immunocytochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Degeneration of heterogeneous systems in the central nervous system, with widespread distribution of argyrophilic neuronal fibrillary inclusions, was found in a patient with presenile dementia. Atrophy was circumscribed in the frontal and temporal lobes. Neuronal loss was severe in the basal ganglia, subthalamic nucleus, and substantia nigra. Immunocytochemical study using anti-phosphorylated tau and anti-ubiquitin antibodies in conjunction with ultrastructural observations revealed two types of inclusions: neurofibrillary tangles (NFTs) of progressive supranuclear palsy (PSP) in the Edinger-Westphal nucleus, locus coeruleus, cerebellar dentate nucleus, inferior olivary nucleus, and posterior horn of the spinal cord; and Pick bodies (PBs) in the atrophied cerebral cortex and red nucleus. PSP-type NFTs and PBs have been demonstrated in a single case for the first time. Despite their pathognomonic significance in certain disorders, we suggest that these inclusions may reflect a form of cytoskeletal disorganization, which is not entirely restricted to a single disease entity.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0533
    Keywords: Localized hypertrophic neuropathy ; Electron microscopy ; Immunohistochemistry ; Nerve growth factor receptor ; Schwann cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Immunoreactivity for nerve growth factor receptor (NGFR) was examined using a monoclonal antibody against human NGFR in the sural nerve of a 24-year-old woman, affected by localized hypertrophic neuropathy (LHN). NGFR expression was correlated with electron microscopy and with immunoreactivity for S-100 protein, laminin, HLA-DR, HNK-1, P0 glycoprotein and neurofilament peptides. Our results indicate that in LHN most of whorl-forming cells are NGFR positive and S-100 protein or HLA-DR negative. These data along with the ultrastructural features suggest their origin from perineurium.
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 186 (1992), S. 407-429 
    ISSN: 1432-0568
    Keywords: Acetylcholine receptors ; Immunocytochemistry ; Electron microscopy ; Human motor endplate ; Human cerebral cortex ; Neurodegenerative disorders
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Acetylcholine and its receptors are involved in a variety of important signal transduction processes. As shown here paradigmatically for the human neuromuscular junction and the cerebral cortex, acetylcholine receptors can be visualized immunohistochemically at the cellular and subcellular level under physiological and pathological conditions. At normal motor endplates nicotinic cholinoceptors are localized at the surface of the postsynaptic junctional folds. In myasthenic syndromes investigation of muscle biopsies enables the diagnosis of receptor deficiencies at the ultrastructural level. In normal cerebral cortex pyramidal neurons are equipped with both nicotinic and muscarinic acetylcholine receptors localized to postsynaptic densities. In neuropsychiatric diseases cholinoceptor expression can be monitored at the cellular level by quantititative assessment of immunolabeled cortical neurons.
    Type of Medium: Electronic Resource
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 185 (1992), S. 547-557 
    ISSN: 1432-0568
    Keywords: Dorsal tegmental nucleus of Gudden ; Laterodorsal tegmental nucleus ; Synaptic organization ; Anterograde tracing study ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We examined the synaptic organization of ascending projections from the pars ventralis of the dorsal tegmental nucleus of Gudden (TDV) and the laterodorsal tegmental nucleus to the lateral mammillary nucleus (LM). The LM neuropil consists of terminals containing pleomorphic synaptic vesicles and forming symmetric synaptic contact, and terminals containing round synaptic vesicles and forming asymmetric synaptic contact. They make up 63% and 37%, respectively, of all axodendritic terminals. All axosomatic terminals contain pleomorphic vesicles and make symmetric contact. Following injection of WGA-HRP into the TDV, many anterogradely labeled terminals and retrogradely labeled cells are found in the LM. Labeled terminals contact mainly proximal (more than 2 μm diameter) and intermediate (1–2 μm diameter) dendrites. Serial ultrathin sections of the LM show that 55% of axosomatic terminals are labeled anterogradely. Following injection of WGA-HRP into the laterodorsal tegmental nucleus, many anterogradely labeled terminals are found in the LM, but no retrogradely labeled cells are present. Labeled terminals contact mainly distal (less than 1 μm diameter) and intermediate dendrites as well as somata. In the LM neurons, 46% of axosomatic terminals are labeled anterogradely. All labeled terminals from these nuclei contain pleomorphic vesicles and make symmetric synaptic contact. These results indicate that almost all axosomatic terminals come from the TDV and the laterodorsal tegmental nucleus, which send inhibitory inputs to the lateral mammillary nucleus.
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 83 (1992), S. 134-139 
    ISSN: 1432-0533
    Keywords: Scrapie ; Creutzfeldt-Jakob disease ; Neuronal autophagy ; Neuropathology ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We report the presence of autophagic vacuoles (AV) in neuronal perikarya and neuronal processes of rodents with experimental scrapie and Creutzfeldt-Jakob disease. AV were composed of sequestrated cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles. The formation of AV may contribute to neuronal degeneration and ultimately to neuronal loss.
    Type of Medium: Electronic Resource
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  • 14
    ISSN: 1432-0533
    Keywords: Alzheimer's disease ; Astrocytes ; Electron microscopy ; Immunohistochemistry ; Paired helical filaments
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A 54-year-old man who had cerebellar ataxia and pseudobulbar palsy at the age of 29 years, and soon developed dementia, myoclonus and convulsions, died after about 20 years in a vegetative state. Histological examination of the extensively atrophic and devastated brain (680 g) revealed the almost total loss of cerebral cortical neurons associated with numerous β-protein amyloid plaques, many extracellular tangles and a large number of hypertrophic astrocytes, and prominent amyloid angiopathy. The astrocytes were frequently immunopositive for anti-human tau antibody (anti-htau) and anti-ubiquitin antibody (anti-ubi). Double immunostaining with anti-htau and anti-glial fibrillary acidic protein (GFAP) antibody clearly demonstrated htau-positive domains within the GFAP-positive perikarya/and processes of several astrocytes. Electron microscopy of the hippocampal CA1, which was completely devoid of pyramidal neurons, revealed, in astrocytes, abnormal filaments indistinguishable from the paired helical filaments (PHFs) seen in neurons. On immunoelectron microscopy, the filaments were observed to be labeled with anti-htau and anti-ubi, exhibiting the same immunohistochemical features as neuronal PHFs. This is the first demonstration of clearly constricted and both tau- and ubiquitin-positive PHFs in astrocytes, indicating that, in some special conditions like in our case, processes similar to those that attack neurons also affect astrocytes and ultimately make the latter form PHFs.
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 186 (1992), S. 363-370 
    ISSN: 1432-0568
    Keywords: Retinal photoreceptors ; Electron microscopy ; Teleost ; Astronatus ocellatus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The structure and arrangement of the retinal photoreceptors of the velvet cichlid fish (Astronotus ocellatus) have been studied by light and electron microscopy. Rods, single cones and double (twin) cones are present. In the light-adapted state, rods are very tall cells that reach deep into the retinal epithelial (RPE) layer. The long outer segment is composed of discs of uniform diameter displaying one or two incisures. The rod inner segment shows a distal ellipsoid of mitochondria, and then narrows dramatically in the myoid region. Rod nuclei are electron dense and located deep in the outer nuclear layer. Rod synaptic spherules are small and show two to three invaginated synaptic sites as well as superficial synapses. Single cones are similar to the individual members of a double cone and all display a short tapering outer segment, a large ellipsoid of mitochondria and a myoid rich in rough endoplasmic reticulum, polysomes, Golgi zones and autophagic vacuoles. Double cones have extensive subsurface cisternae along their entire contiguous surfaces. Cone nuclei are large and vesicular and located close to or through the external limiting membrane. The synaptic pedicles of cones are larger, more electron lucent, and display more invaginated (ribbon) synapses as well as conventional (superficial) synaptic sites than do the rod spherules. Rod photoreceptors certainly undergo retinomotor movements and it is probable that cones do as well. In the light-adapted state the cone photoreceptors are arranged in a repeating square mosaic pattern with one single cone surrounded by four double (twin) cones.
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 84 (1992), S. 630-637 
    ISSN: 1432-0533
    Keywords: Dentrites ; Excitotoxicity ; Glutamate ; N-Methyl-d-aspartate ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary This study documents the ultrastructural features of acute neuronal injury following N-methyl-d-aspartate (NMDA) receptor activation. NMDA (100 nmol/μl) or vehicle was infused over a 15-min period into the lateral ventricle of adult rats. After perfusion fixation, specimens demonstrating normal and abnormal patterns of vascular permeability to horserdish peroxidase were sampled for ultrastructural analysis. In NMDA-infused rats, brain regions exhibiting protein extravasation contained swollen dendritic profiles and abnormal neuronal perikarya. Although periventricular regions were most severely affected, parenchymal abnormalities were also detected in the cerebral cortex, septum, striatum, thalamus, hypothalamus and cerebellum. Mildly affected dendrites contained dark compact mitochondria, while in severely swollen dendrites mitochrondia were enlarged with ruptured cristae. Focal sites of plasma membrane disruption were also detected within swollen dendrites. Swollen neurons commonly displayed peripheral pallor and increased numbers of cytoplasmic vacuoles. Other neurons appeared dark and shrunken, some containing disrupted mitochrondria and pyknotic nuclei. Pretreatment with the NMDA antagonist MK-801 (2 mg/kg) attenuated the neuronal and dendritic alterations. In conditions where cerebrospinal fluid levels of glutamate are abnormally elevated, excessive NMDA receptor activation may lead to early vascular and neuronal complications which could work in concert to promote brain injury.
    Type of Medium: Electronic Resource
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 84 (1992), S. 621-629 
    ISSN: 1432-0533
    Keywords: Blood-brain barrier ; Endothelium ; Excitotoxicity ; N-Methyl-d-aspartate ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The purpose of this study was to document the early cerebrovascular consequences of excessive N-methyl-d-aspartate (NMDA) receptor activation. Five microliters of NMDA (100 nmol/μl) or vehicle was infused over a 15-min period into the lateral ventricle of adult rats. The protein tracer horseradisch peroxidase (HRP) was injected intravenously for blood-brain bartier (BBB) studies. The intraventricular infusion of vehicle (n=5) caused no alterations in arterial blood pressure or microvascular damage away from the intraventicular probe tract. In contrast, NMDA infusion (n=8) led to a gradual increase in arterial blood pressure (mean 36 mm Hg). Multifocal regions of HRP extravasation were observed bilaterally throughout the neuraxis following NMDA infusion. Sites of BBB disruption and hemorrhage included brain regions bordering ventricular spaces. In addition, isolated foci of protein extravasation were commonly detected in the cerebral cortex, thalamus, basal forebrain, septum and cerebellum. Pretreatment with the noncompetitive NMDA antagonist MK-801 (2 mg/kg) substantially reduced the BBB responses to NMDA. However, microvascular abnormalities were seen in NMDA-infused rats where blood pressure elevations were inhibited by blood removal. In addition to neurons, cerebral blood vessels are also acutely affected by NMDA receptor activation. Blockage of NMDA receptor channels following brain injury may potentially provide protection by attenuating BBB breakdown and subsequent brain edema.
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  • 18
    ISSN: 1432-0533
    Keywords: Striatonigral degeneration ; Putaminal pigment ; Iron ; Atomic absorption spectroscopy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We measured the total content of iron, cooper, zinc and manganese in the putamen of four patients with striatonigral degeneration (SND) and age-and gender-matched normal controls. The iron content in the SND patients was five times greater than in the controls. Electron microscopic histochemistry revealed the iron reaction products in the pigments showing a triphasic pattern of coarse, electron-dense globules, fine granular and fibrillary materials, and lamellated structures. These findings suggest that increased iron deposition may be related to pigment formation in the putament of SND.
    Type of Medium: Electronic Resource
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  • 19
    ISSN: 1432-0533
    Keywords: Histiocytic markers ; Astrocytoma ; Electron microscopy ; Glial fibrillary acidic protein ; Granular cell tumor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A case of cerebral granular cell tumor (GCT) is reported. Histologically, the growth was composed of benign astrocytes, granular cells and transitional forms between both elements. Glial fibrillary acidic protein was detected in the glial component and, to a lesser extent, in the granular cells. Alpha-1-antichymotrypsin was demonstrated in the latter component only. Ultrastructural study also supported the evidence that neoplastic astrocytes became granular cells. The survey of the literature and our own results suggest that GCTs in this particular location, even when histologically benign, seem to have a worse prognosis than the low-grade supratentorial astrocytomas.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 186 (1992), S. 371-377 
    ISSN: 1432-0568
    Keywords: Retinal pigment epithelium (RPE) ; Electron microscopy ; Teleost ; Astronatus ocellatus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The morphology of the retinal pigment epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) have been studied by light and electron microscopy in the velvet cichlid (Astronotus ocellatus). The RPE is composed of a single layer of large columnar cells. The basal (scleral) border of these cells is minimally infolded, whereas the apical (vitreal) surface displays numerous pigment-laden processes which in light-adaptation surround both rod and cone outer segments. Laterally the RPE cells are joined by a series of basally located tight junctions. Wandering phagocytes are a constant feature within this epithelial membrane. The RPE cells display a large, vesicular nucleus, numerous mitochondria, much smooth endoplasmic reticulum, polysomes, myeloid bodies, phagosomes and melanosomes. Rough endoplasmic reticulum is relatively scarce within these cells. Although only light-adapted specimens were examined, it is thought that the melanosomes are capable of extensive retinomotor movement. The endothelium of the choriocapillaris facing Bruch's membrane is typically very thin but shows few fenestrations. Bruch's membrane is typical of other teleost species in that it is composed of only three layers.
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  • 21
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 186 (1992), S. 519-527 
    ISSN: 1432-0568
    Keywords: Intestine ; Interstitial cells ; Innervation ; Electron microscopy ; Guinea pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Systematic examination using electron microscopic montages and serial sections has demonstrated that three types of interstitial cell, namely gap junction-rich cells, glycogen-rich cells and fibroblast-like cells, are densely located along the whole extent of the deep muscular plexus of the guinea pig small intestine. They tend to be distributed in an alternating fashion in the cellular network, connected with muscle cells of the outer, circular layer by means of gap junctions. These three types of interstitial cell show close relations to two types of nerve varicosity: one type is characterized by clear round vesicles with diameters of about 50 nm, and the other by flattened vesicles measuring about 35 nm by 70 nm. Electron-dense patches have been observed at the cytoplasmic side of the axonal membranes. Muscle cells of both inner and outer circular layers also show close relations to these two types of nerve varicosity. These morphological features are discussed with the implication that they may have some regulatory role in intestinal movement.
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  • 22
    Electronic Resource
    Electronic Resource
    Springer
    Archives of dermatological research 284 (1992), S. 290-296 
    ISSN: 1432-069X
    Keywords: Cepharanthine ; Minoxidil ; Culture ; Hair cells ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The effects of cepharanthine and minoxidil on proliferation, differentiation and keratinization of cultured cells from the murine hair apparatus were examined electron microscopically. Both cepharanthine and minoxidil stimulated cell proliferation and delayed initiation of differentiation and keratinization of the cultured cells. On day 6, most control cells (87%) cultured in a 0.03 mM calcium medium without cepharanthine and minoxidil were differentiated into several subpopulations corresponding to those of in vivo cell layers of the hair apparatus, while most of the cells cultured with cepharanthine (71%) or minoxidil (70%) were still immature. On day 13, the number of degenerated cells increased (63%) in the control culture, whereas in the culture treated with cepharanthine or minoxidil, cell degeneration scarcely occurred (5% and 8%, respectively). Differentiated cells having tonofilaments were often observed in the cepharanthine- and minoxidil-treated cultures (76% and 72%, respectively). Elevation of extracellular calcium up to 1.0 mM induced keratinization (34%) in the control culture on day 6, while no keratinized cells were observed in the cepharanthine- or minoxidil-treated culture. On day 13 keratinization similarly occurred in the cultures with cepharanthine (30%) or minoxidil (48%). These results show that both cepharanthine and minoxidil may directly influence proliferation, differentiation and keratinization of cultured cells from the hair apparatus.
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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    Archives of dermatological research 284 (1992), S. 242-245 
    ISSN: 1432-069X
    Keywords: Immunogold ; Electron microscopy ; Human skin ; Actin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Normal human skin was embedded in Lowicryl K4M. Actin microfilaments were localized by applyinga postembedding immunogold technique using the monoclonal anti-actin antibody HHF35. Actin microfilaments are part of the cytoskeleton in muscle and nonmuscle cells. Together with myosin they produce contraction. The antibody labelled myofilaments in smooth muscle arrector pili cells, myoepithelial cells and pericytes. In sweat gland cells the microvilli system, a zone beneath the cytoplasma membrane correponding to the adhesion belt region, and apocrine decapitation formations showed labelling.
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  • 24
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    Archives of dermatological research 284 (1992), S. 303-306 
    ISSN: 1432-069X
    Keywords: Hair follicle ; Basal lamina ; Dermal papilla cells ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 25
    ISSN: 1432-1238
    Keywords: Catheter ; Electron microscopy ; Infection ; Adherence ; Candida albicans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Objective The purpose of the study was do describe the architecture of accretions occurring on the tips of central venous catheters (CVC). Design A conservative procedure was used followed by two different techniques of electron microscopy Setting and patients the study included 19 catheters which have been used on intensive cared adults, and which were chosen among those of parallel 300 CVC study. Measurements and results CVC were considered sterile, contaminated, colonized or infected according to microbiological and clinical criteria. CVC were found to remain much cleaner than in past clescriptions. When present, accretions were located on the olive-shaped end, and displayed stratified structures with three types of material: amorphous material, thrombus components and inflammatory cells. Bacteria were not seen, even on culture positive CVC. Candida albicans was found on one CVC in the cytoplasm of ganulocytes, and made xio direct contact with the plastic surface. Conclusion This technique should contribute to the understanding of the pathobiology of CVC infection and provide information proving or precluding the involvement of microbial adherence to polymers in vivo.
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  • 26
    ISSN: 1432-1106
    Keywords: Immunocytochemistry ; Visual pathways ; Electron microscopy ; Hamster
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The expression of specific calcium binding proteins is being used increasingly as a potential neuroanatomical marker for neurons with similar functions. In this study, the distribution of calbindin D28k in the superior colliculus (SC) of adult hamsters was examined by light and electron microscopy. Calbindin immunoreactivity was prominent in specific regions and laminae of the SC throughout its rostrocaudal extent, and was found to label horizontal, vertical and stellate cell types. In addition, calbindin label highlighted “bridges” of neuronal processes in the intermediate layers. The most frequent calbindin-immunoreactive profiles seen in the electron microscope were dendrites, some of which were post-synaptic to apparent retinal ganglion cell axon terminals. Labelled axons and axon terminals were less frequently encountered. There was considerable overlap between the size distribution of calbindin D28k-immunoreactive neurons and that of GABA-immunoreactive or Nissl stained neurons in the SC. However, using a double fluorescent labelling technique, and examination of the tissue with confocal laser microscopy, no neurons were observed in the hamster SC that showed immunoreactivity for both calbindin and GABA. In this regard, the SC is similar to the mammalian lateral geniculate nucleus and the pretectum, but differs from the neocortex, where calbindin and GABA are colocalized. The demonstration in the SC, as well as other parts of the nervous system, of sub-populations of neurons that contain distinct calcium-binding proteins suggests that these neurons have different functional properties. Correlative studies may clarify the relevance of these cytoplasmic components as cell markers, as well as their different patterns of association with neurotransmitters and peptides.
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  • 27
    ISSN: 1432-2048
    Keywords: Bradyrhizobium ; Electron microscopy ; Glycine (root nodules) ; High-pressure freezing ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract High-pressure freezing of chemically untreated nodules of soybean (Glycine max (L.) Merr.), in sharp contrast to chemical fixation and prefixation, appears to preserve the ultrastructure close to the native state. This is supported by the observation that the peribacteroid membrane of high-pressure-frozen samples is tightly wrapped around the bacteroids, a finding that is fully consistent with the current views on the physiology of oxygen and metabolite transport between plant cytosol and bacteroids. In soybean root nodules, the plant tissue and the enclosed bacteria are so dissimilar that conventional aldehyde-fixation procedures are unable to preserve the overall native ultrastructure. This was demonstrated by high-pressure freezing of nodules that had been pre-fixed in glutaraldehyde at various buffer molalities: no buffer strength tested preserved all ultrastructural aspects that could be seen after high-pressure freezing of chemically untreated nodules.
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  • 28
    ISSN: 1432-0738
    Keywords: Bis(tributyltin) oxide ; Liver ; Electron microscopy ; X-ray microanalysis ; Biochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.
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  • 29
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    Archives of microbiology 157 (1992), S. 381-388 
    ISSN: 1432-072X
    Keywords: PhiX174 ; Bacterial lysis ; Escherichia coli ; Electron microscopy ; Membranes ; Cell envelope
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.
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  • 30
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    Child's nervous system 8 (1992), S. 326-331 
    ISSN: 1433-0350
    Keywords: Medulloblastomas ; Immunohistochemistry ; Cytokeratins ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Thirty-five paraffin-embedded medulloblastomas (19 from children and 16 from adults; 24 classic medulloblastomas, 10 desmoplastic medulloblastomas, 1 tumor with neuronal differentiation) were examined for reactions with antibodies against glial fibrillary acidic protein (GFAP), cytokeratins KL1 and MNF116, desmin, and vimentin. Only the tumor from the youngest patient, a 152-day-old boy, showed a positive immunoreaction for cytokeratins. Because of this age-related expression of cytokeratins in medulloblastomas primarily in very young children, cytokeratin positivity was interpreted as a sign of tumor immaturity. Five medulloblastomas showed scattered GFAP-positive reactive astrocytes and/or other positive, probably neoplastic, cells. Only two tumors showed GFAP immunoreactivity in unequivocally neoplastic cells. Of six tumors that reacted with vimentin, three showed strong reactivity throughout, one being the tumor from the 152-day-old boy. The remaining three demonstrated nests of vimentin-positive cells with weak or intense somatic immunoreactivity for vimentin. None of the 35 cases showed positivity for desmin, indicating that mesenchymal differentiation is restricted to the rare so-called medullomyoblastomas.
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  • 31
    ISSN: 1573-4935
    Keywords: Transferrin ; Receptor ; Isolation ; Reconstitution ; Liposomes ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptortransferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon, detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w) Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed ographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5×10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2–3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.
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  • 32
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    Bioscience reports 12 (1992), S. 495-501 
    ISSN: 1573-4935
    Keywords: Electron microscopy ; secretion ; neuropeptides ; exocytosis ; endocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.
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  • 33
    ISSN: 1432-2072
    Keywords: Haloperidol ; Clozapine ; Perforated synapses ; Electron microscopy ; Tardive dyskinesia ; Extrapyramidal syndrome
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Perforated synapses, which contain a discontinuous density along the postsynaptic membrane, can increase or decrease in numbers following various behavioral and biochemical manipulations. We have previously established that 14-day treatment with haloperidol causes an increase in the number of perforated synapses within the caudate nucleus (dorsolateral region) but not the nucleus accumbens (Meshul and Casey 1989). This effect was reversed if the animals were withdrawn from the drug for an equivalent period of time. We have now further examined the effects of haloperidol administration, which is associated with a high incidence of extrapyramidal side effects (EPS) and tardive dyskinesia (TD), and assessed the effects of clozapine, which appears to have a lower potential for inducing EPS and TD. Administration of haloperidol for 2 weeks significantly increased the percentage of perforated synapses in the caudate, but not in the nucleus accumbens or layer VI of medial prefrontal cortex (MPCx). There was an increase in specific [125I]epidepride binding to D-2 receptors in the caudate nucleus and MPCx following haloperidol. Administration of clozapine for 2 weeks did not affect the percentage of perforated synapses in any of the three dopamine (DA)-rich regions that were examined. There was an increase in specific [3H]SCH 23390 binding to D-1 receptors and in specific [125I]epidepride binding to D-2 receptors only within MPCx following clozapine. The absence of any change in the density of perforated synapses within the dorsolateral caudate nucleus following clozapine correlates with: 1) the lack of effect on specific DA receptor binding or down regulation of serotonin (5-HT2) receptors (as reported by others), or 2) the inability in clozapine-treated animals to depolarize block substantia nigra (A9) DA neurons. These results may be related to the low incidence of EPS and TD observed with clozapine.
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  • 34
    ISSN: 1432-0878
    Keywords: Chordotonal organ ; Scolopidium ; Homology ; Electron microscopy ; Sensilla ; Evolution ; Actias luna (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The structure of a simple chordotonal organ, the presumed homologue of the noctuoid moth tympanal organ, is described in the atympanate moth, Actias luna. The organ consists of a proximal scolopidial region and a distal strand, which attaches peripherally to the membranous cuticle ventral to the hindwing alula. The strand is composed of elongate, microtubule-rich cells encased in an extracellular connective tissue sheath. The scolopidial region houses three mononematic, monodynal scolopidia, each comprised of a sensory cell, scolopale cell, and attachment cell. The dendritic apex is octagonally shaped in transverse section, its inner membrane lined by a laminated structure reminiscent of the noctuoid tympanal organ ‘collar’. A 9+0-type cilium emerges from the dendritic apex, passes through both the scolopale lumen and cap, and terminates in an extracellular space distal to the latter. Proximal extensions of the attachment cell and distal prolongations of the scolopale cell surrounding the cap are joined by an elaborate desmosome, with which is associated an extensive electron-dense fibrillar plaque. Within the scolopale cell, this plaque constitutes the scolopale ‘rod’ material. The data are discussed in terms of both the organ's potential function, and its significance as the evolutionary proto-type of the noctuoid moth ear.
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  • 35
    ISSN: 1432-0878
    Keywords: Retina ; Dopaminergic neurons ; Synapses ; Inner plexiform layer ; Immunocytochemistry ; Electron microscopy ; Bufo marinus (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Tyrosine hydroxylase (TH) immunocytochemistry was utilized to quantify dopaminergic synapses in the inner plexiform layer of the retina of Bufo marinus. Since dopaminergic cells have bistratified dendritic arborisation in the inner plexiform layer, attention was given to the segregation of synapses between the scleral and the vitreal sublaminae. Light-microscopically, a more elaborate dendritic branching was observed in the scleral than in the vitreal sublamina. In contrast, about 55% of synapses occurred in the vitreal one fifth of the inner plexiform layer, 30% in the scleral fifth, and 15% in the intermediate laminae. Input sources and output targets showed only minor quantitative differences between sublaminae 1 and 5. TH-immunoreactive processes were found in presynaptic (62.8%) and postsynaptic (37.2%) positions. Synapses to the stained dendrites derived from bipolar (40.4%) and amacrine (59.6%) cells, whereas outputs from the TH-positive processes were directed to amacrine cells (56.8%) and to small and medium-sized dendrites (35.4%); at least some of these can be considered as ganglion cell dendrites. TH-positive profiles neither formed synapses with each other nor were presynaptic to bipolar cell terminals. Junctional appositions of the immunoreactive profiles were occasionally seen on non-stained amacrine and ganglion cell dendrites in the scleral sublamina of the inner plexiform layer and on optic axons in the optic fibre layer. Although dopaminergic cells are mainly involved in amacrine-amacrine interactions, inputs from bipolar terminals and outputs to ganglion cell dendrites were also substantial, suggestive of a role also in vertical information processing.
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  • 36
    ISSN: 1432-0878
    Keywords: Regeneration ; Skeletal muscle ; Injury ; Autoradiography ; Morphometry ; Electron microscopy ; Mouse (SJL/J BALB/c)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.
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  • 37
    ISSN: 1432-0878
    Keywords: Enkephalins ; Neuropeptides ; Neurohemal organ ; Immunogold ; Electron microscopy ; Carcinus maenas, Uca pugilator, Eriocheir sinensis (Crustacea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.
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  • 38
    ISSN: 1432-0878
    Keywords: Basic fibroblast growth factor ; Regeneration ; Degeneration ; Immunohistochemistry ; Electron microscopy ; Masseter muscle ; Myoneural junction ; Mouse (dystrophic mdx)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The localization of basic fibroblast growth factor (bFGF)-like immunoreactivity in the masseter muscle of dystrophic mdx mice on postnatal day 28 was investigated by immunoblot analysis and electron microscopy. Crude homogenate of the masseter muscle, when subjected to immunoblotting with a bFGF antiserum, exhibited a main band with the same molecular weight (18 kDa) as bovine bFGF. By electron microscopy, bFGF immunoreactivity was detected in small regenerating myocytes; the smaller cells were the premature myocytes, the most intense staining was the immunoreactivity within the cytoplasm. Putative precursors of the muscle cells with a few myofilaments, which were most intensely labeled with anti-bFGF, contacted each other and possibly developed into multinucleated myocytes through cell fusion. Mature myocytes with densely packed myofilaments and peripherally located nuclei did not exhibit bFGF immunoreactivity; they formed myoneural junctions with motor nerve endings immunoreactive for bFGF. Early differentiating myocytes with intense bFGF-like immunoreactivity did not make contact with immunoreactive nerve terminals. Degenerating large myocytes with a limited number of distorted and/or disrupted myofilaments exhibited electron-dense deposits in the cristae of mitochondria; these deposits were not abolished by immunoadsorption control experiments. Thus, the cell-size-dependent decrease in bFGF immunoreactivity in regenerating but not in degenerating myocytes provides a morphological basis for an autoregulatory role of bFGF in muscle regeneration. This study suggests that neuronal bFGF is not involved in initial muscle regeneration in the dystrophic mdx mouse.
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  • 39
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    Cell & tissue research 270 (1992), S. 87-93 
    ISSN: 1432-0878
    Keywords: Ovarian nerves ; Development ; Folliculogenesis ; Tyrosine hydroxylase ; Immunohistochemistry ; Electron microscopy ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.
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  • 40
    ISSN: 1432-0878
    Keywords: Adrenal cortex ; Ageing ; Steroidogenesis ; Electron microscopy ; Morphometry ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.
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  • 41
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    Cell & tissue research 270 (1992), S. 273-279 
    ISSN: 1432-0878
    Keywords: Macrophages ; Small intestine ; Large intestine ; External muscle layer ; Immunohistochemistry ; Histochemistry ; Electron microscopy ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electronmicroscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.
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  • 42
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    Cell & tissue research 267 (1992), S. 321-335 
    ISSN: 1432-0878
    Keywords: Teeth ; Cementum ; Cementoblasts ; Matrix production ; Electron microscopy ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The present study describes the formative process of the initiation of cellular intrinsic fiber cementum (CIFC) in still growing human teeth. From 29 premolars and molars with incomplete roots developed to 60–90% of their final length, 8 premolars (with roots formed to three quarters of their final length) were selected for electron-microscopic investigation. All teeth were clinically intact and prefixed in Karnovsky's fixative immediately after extraction. Most of them were decalcified in ethylene diaminetetraacetic acid (EDTA), and the apical part of the roots was divided axially into mesial and distal portions that were subdivided in about 5 slices each. Following osmication and embedding in Epon, these blocks were cut for light- and electron-microscopic examination. In addition, 5 teeth with incomplete roots were freed from organic material and processed for scanning electron microscopy. It was found that CIFC-initiation commenced very close to the advancing root edge and resulted in a rapid cementum thickening. Thereafter, appositional growth continued on the already established cementum surface. Large, basophilic and rough endoplasmic reticulum-rich cementoblasts, some of which became cementocytes, were responsible for both fast and slow CIFC-formation. The CIFC-matrix was free of Sharpey's fibers and composed of more or less organized intrinsic collagen fibrils, in part fibril bundles, that ran roughly parallel to the root surface. Initially, the cementum fibrils intermingled with those of the dentinal collagen fibrils, which were not yet mineralized. This boundary subsequently underwent calcification. The development of collagen fibril bundles and their extracellular arrangement were associated with cytoplasmic processes probably involved in fibril formation and fibril assembly. Many cementoblasts contained intracytoplasmic, membrane-bounded collagen fibrils, which probably were related to fibril formation rather than degradation.
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  • 43
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    Cell & tissue research 267 (1992), S. 385-389 
    ISSN: 1432-0878
    Keywords: Leydig cells ; Macrophages ; Development, ontogenic ; Electron microscopy ; Testis ; (Rat Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.
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  • 44
    ISSN: 1432-0878
    Keywords: Intestine ; Interstitial cells ; Pacemaker ; Electron microscopy ; Guinea-pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine were studied by electron microscopy, and three-dimensional cell models were reconstructed from serial ultrathin sections with a computer graphic system. Three types of cells were recognized. The first type was similar in shape to smooth muscle cells, but did not contain an organized contractile apparatus. Many large gap junctions comprising about 4% of the cell surface were present; they connected cells of the first type to each other, to the second type of cell and to smooth muscle cells of the outer circular layer. The second type of cell had a welldemarcated cell body with long slender processes and was characterized by a large amount of glycogen comprising about 9% of the cell volume. The third type of cell was similar to fibroblasts, and contained well-developed Golgi apparatus and rough endoplasmic retiulum. Some of these fibroblast-like cells (a possible subtype) formed small gap junctions. All three types of cells showed close relationships with nerve varicosities. This cellular network consisting of gap-junction-rich cells, glycogen-rich cells and smooth muscle cells may be involved in the pacemaking activity of intestinal movement.
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  • 45
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    Cell & tissue research 268 (1992), S. 239-245 
    ISSN: 1432-0878
    Keywords: Urinary bladder ; Epithelial desquamation ; TPA ; Electron microscopy ; Rat (Donryu)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1μg/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.
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  • 46
    ISSN: 1615-6102
    Keywords: ATPase ; UTPase ; Electron microscopy ; Energy-dispersive X-ray microanalysis ; Gomphrena globosa ; Histochemistry ; Hordeum distichon ; Monstera deliciosa ; Phloem
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary For the histochemical localization of nucleoside triphosphatases at the electron microscopic level, prefixed tissues were incubated with lead nitrate in addition to substrate (GOMORI reaction). While ATP and UTP as substrates gave electron-dense reaction products at the plasmalemma of sieve tubes, companion cells and phloem parenchyma cells, and at plasmodesmata in primary pitfields, AMP gave reaction products only at the tonoplast of parenchyma cells. Since electron-dense deposits also occur in cell walls and vacuoles, energy dispersive X-ray microanalysis was used to distinguish between lead deposits and lead-phosphate deposits. The latter were restricted to the symplast. Among the three plant species used, the leaf bundle phloem ofHordeum distichon showed ATPase activity largely restricted to the phloem cells, except for the thickwalled sieve tubes. Some activity also bordered the chloroplasts of the bundle sheath cells. In the C4 plantGomphrena globosa, ATPase and UTPase activities appeared to be the greater in phloem parenchyma cells than in sieve tubes. In the phloem of youngMonstera deliciosa roots, ATPase occurred not only at the plasmalemma of sieve tubes, but also around sieve-tube plastids. When compared with AMP as substrate, it appears that nucleoside triphosphates are the natural substrates of the enzyme(s) in the plasmalemma of sieve tubes and phloem parenchyma cells.
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  • 47
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    European archives of oto-rhino-laryngology and head & neck 249 (1992), S. 168-171 
    ISSN: 1434-4726
    Keywords: Cochlea-Stria vascularis ; Electron microscopy ; Endocochlear potential ; Loop diuretics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The purpose of this study was to compare the effects of several organic acids (probenecid, sodium salicylate and penicillin G) on the endocochlear potential (EP) and the ultrastructure of the stria vascularis of the chinchilla with the effects of furosemide on these parameters. chinchillas received 50 mg/kg i.v. doses of probenecid, sodium salicylate or penicillin G, or 25 mg/kg i.v. furosemide. The EP was monitored continuously before and for 60 min afterwards. The stria vascularis was removed at 10-min intervals from animals and from 10 to 60 min after the injection of these agents. Specimens were then processed for transmission electron microscopy. Only furosemide had an effect on the EP, causing a reversible reduction. The reduction of the EP was accompanied by the appearance of edema in the intercellular spaces of the stria vascularis. No significant edema was found after probenecid, sodium salicylate or penicillin G. This was consistent with the finding that none of these latter three agents affected the endocochlear potential.
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  • 48
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    European archives of oto-rhino-laryngology and head & neck 249 (1992), S. 216-223 
    ISSN: 1434-4726
    Keywords: Cricopharyngeal dysphagia ; Muscle biopsy ; Electron microscopy ; Enzyme histochemistry ; Myositis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The structural changes in the cricopharyngeal muscle (CM) were examined ultrastructurally and by enzyme histochemistry in five patients suffering from idiopathic cricopharyngeal dysphagia (ICD). Diagnosis was established by fiberoptic esophagoscopy, esophageal manometry and cineradiography. Cricopharyngeal myotomy was performed with marked improvement in all patients. Intraoperatively, a biopsy was taken from the CM. Additionally, all patients underwent neurological examination for possible generalized muscle disease, and a biopsy was taken from a limb muscle. CM from nine cadavers without known history of dysphagia served as control. The control samples disclosed structural changes which were considered to be pathological in other skeletal muscles, and required that the criteria for CM pathology we modified accordingly. In three patients changes in CM histology suggested specific pathogenesis: one patient had evidence for a generalized myositis but was only symptomatic for dysphagia. Another patient had muscle fiber atrophy and slight inflammation in her CM, possibly due to alcohol abuse. The third patient had loss of CM fibers with replacement by connective tissue enough to cause functional disturbances. In two patients no cause for dysphagia was found in either immunohistochemistry or electron microscopic studies. These results demonstrate the special structural features of the CM and indicate that ICD can have multiple etiologies.
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  • 49
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    European archives of oto-rhino-laryngology and head & neck 249 (1992), S. 224-230 
    ISSN: 1434-4726
    Keywords: Inner ear ; Rat spiral limbus ; Intercellular matrix ; Electron microscopy ; Immunomorphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The matrix of the spiral limbus is obviously a special form of the intercellular substance. In the present study, the rat's spiral limbus was investigated by electron microscopy after fixation with ruthenium red and tannic acid and immunofluorescence to demonstrate matrix components. Collagen types I, II, V, VI, VII, IX and XI and fibronectin were not observed. Collagen type II and cartilage-specific proteoglycans, however, occurred in large quantities. The basal lamina of interdental cells and inner sulcus cells did not contain any collagen type IV. while the basal lamina of the capillaries had only minor amounts. Laminin and nidogen appeared in large amounts in the basal lamina. After fixation with tannic acid, the matrix between the interdental cells and the capillaries contained 20- to 22-nm-thick single and irregularly running fibrils as well as plaques of a fine granular material. After fixation with ruthenium red, 30-to 60-nm-thick, electron-dense granules occurred and most probably consisted of proteoglycans. These findings indicate that the composition of the matrix of the spiral limbus is similar to that of cartilage but not identical.
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  • 50
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    European archives of oto-rhino-laryngology and head & neck 249 (1992), S. 287-292 
    ISSN: 1434-4726
    Keywords: Nerve endings ; Outer hair cells ; Acoustic exposure ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Rabbits were exposed to 100 dB pure-tone sound at 2 kHz for 2 h. Electron microscopy was used to study the relationship between changes in the outer hair cells, afferent nerve endings and efferent nerve endings. There was no relationship found between the degree of changes produced in the afferent nerve endings and that the outer hair cells. However, there was a relationship demonstrable between the degree of changes seen in the efferent nerve endings and the infranuclear region of the outer hair cells. These findings show that acoustic trauma will damage most the infranuclear region of the outer hair cells, while efferent nerve endings are injured next and least affected are afferent nerve endings.
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  • 51
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 111-116 
    ISSN: 0887-3585
    Keywords: protein folding ; multiple minima problem ; peptide conformation ; energy calculation ; helices ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to α-helical conformations. The α-helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectroscopy.
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  • 52
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    Proteins: Structure, Function, and Genetics 12 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 53
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 324-330 
    ISSN: 0887-3585
    Keywords: X-ray diffraction analysis ; hydrogen bonds ; peptide conformation ; 310/α-helix transition ; antiparallel helix packing ; leucyl-leucyl interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5→1 hydrogen bonds and two 4→1 hydrogen bonds near the C terminus. Three head-to-tail NH ċ O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ċ Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P212121 with Z = 4 and cell parameters a = 16.774(3) Å, b = 20.032(3) Å and c = 20.117(3) Å; overall agreement factor R = 10.7% for 2014 data with |Fobs| 〈 3σ(F); resolution 1.0 Å.
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  • 54
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 339-344 
    ISSN: 0887-3585
    Keywords: protein folding ; pro region ; protease inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: α-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.
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  • 55
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    Proteins: Structure, Function, and Genetics 13 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 56
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 1-25 
    ISSN: 0887-3585
    Keywords: aspartic proteinase zymogen ; molecular replacement ; structure-function ; activation peptide ; acid activation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 Å. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of β-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (IP-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family. © 1992 Wiley-Liss, Inc.
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  • 57
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    Proteins: Structure, Function, and Genetics 13 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 58
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 70-85 
    ISSN: 0887-3585
    Keywords: major histocompatibility complex ; antigenic peptide ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall α-helical conformation through regular i,i+4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i+3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its α-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts α-helical conformation for the bound peptide. © 1992 Wiley-Liss, Inc.
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  • 59
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 112-119 
    ISSN: 0887-3585
    Keywords: analytical affinty chromatography ; self-association ; HIV p24gag ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution undernonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 × 10-5 M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 × 10-5 M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly. © 1992 Wiley-Liss, Inc.
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  • 60
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 132-140 
    ISSN: 0887-3585
    Keywords: protein stability ; insertion mutations ; substitution mutations ; guanidine hydrochloride denaturation ; conformational changes ; circular dichroism spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:29-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax. From the many alternative packing and bonding arrangements thus made available to the polypeptide chain, the energetically most favorable is selected. © 1992 Wiley-Liss, Inc.
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  • 61
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 213-223 
    ISSN: 0887-3585
    Keywords: protein folding ; protein structure ; rotamers ; simulated annealing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An unknown protein structure can be predicted with fair accuracy once an evolutionary connection at the sequence level has been made to a protein of known 3-D structure. In model building by homology, one typically starts with a backbone framework, rebuilds new loop regions, and replaces nonconserved side chains. Here, we use an extremely efficient Monte Carlo algorithm in rotamer space with simulated annealing and simple potential energy functions to optimize the packing of side chains on given backbone models. Optimized models are generated within minutes on a workstation, with reasonable accuracy (average of 81% side chain χ1 dihedral angles correct in the cores of proteins determined at better than 2.5 Å resolution). As expected, the quality of the models decreases with decreasing accuracy of backbone coordinates. If the backbone was taken from a homologous rather than the same protein, about 70% side chain X1 angles were modeled correctly in the core in a case of strong homology and about 60% in a case of medium homology. The algorithm can be used in automated, fast, and reproducible model building by homology. © 1992 Wiley-Liss, Inc.
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  • 62
    ISSN: 0887-3585
    Keywords: protein structure ; protein sequences ; protein design de novo ; protein engineering ; computer algorithms ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded β-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Fingerclasp, a dimer of interdigitating β-β-α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather - a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.
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  • 63
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 145-157 
    ISSN: 0887-3585
    Keywords: thermal diffuse X-ray scattering ; protein disorder ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Diffuse scattering data have been collected on two crystal forms of lysozyme, tetragonal and triclinic, using synchrotron radiation. The observed diffraction patterns were simulated using an exact theory for simple model crystals which relates the diffuse scattering intensity distribution to the amplitudes and correlations of atomic movements. Although the mean square displacements in the tetragonal form are twice that in the triclinic crystal, the predominent component of atomic movement in both crystals is accounted for by short-range coupled motions where displacement correlations decay exponentially as a function of atomic separation, with a relaxation distance of ≈ 6 Å. Lattice coupled movements with a correlation distance ≈ 50 Å account for only about 5-10% of the total atomic mean square displacements in the protein crystals. The results contradict various presumptions that the disorder in protein crystals can be modeled predominantly by elastic vibrations or rigid body movements.
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  • 64
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 180-187 
    ISSN: 0887-3585
    Keywords: fibronectin ; domain ; collagen ; folding ; disulfide ; fluorescence ; GdmCl ; renaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I “finger” modules and two type II “kringle-like” modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.
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  • 65
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 278-298 
    ISSN: 0887-3585
    Keywords: folding nucleation ; hydrophobic cluster ; conserved loop length ; structure-sequence relationship ; sequence patterns ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Greek key β-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key β-barrel, which forms the core of the third domain of this protein. The hemocyanin β-barrel was found to be structurally very similar to the β-barrels of the immunoglobulin domains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumor proteins. The structural similarity within this group of protein families is not accompanied by an evolutionary or functional relationship. It is therefore possible to study structure-sequence relations without bias from nonstructural constraints. The present study reports a conserved pattern of features in these Greek key β-barrels that is strongly suggestive of a folding nucleation site. This proposed nucleation site, which we call a “β-zipper,” shows a pattern of well-conserved, large hydrophobic residues on two sequential β-strands joined by a short loop. Each β-zipper strand is near the center of one of the β-sheets, so that the two strands face each other from opposite sides of the barrel and interact through their hydrophobic side chains, rather than forming a hydrogen-bonded β-hairpin. Other protein families with Greek key β-barrels that do not as strongly resemble the immunoglobulin fold - such as the azurins, plastocyanins, crystallins, and prealbumins - also contain the β-zipper pattern, which might therefore be a universal feature of Greek key β-barrel proteins.
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  • 66
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 314-323 
    ISSN: 0887-3585
    Keywords: structure analysis ; graph theory ; protein structure ; β sheet ; retrieval ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In order to find rules for the secondary structure prediction of proteins which describe the (sequentially) long-range interactions in sheet structures methods of applied graph theory were used. The so called β graph which describes the sheet topology was defined for every protein in the Brookhaven Data Bank containing β sheets. The resemblance of proteins at that topological level is discussed, and four notations and graphic representations of sheets which describe the sequential and topological neighborhoods of the strands were derived. This description level supports the usage of data structures which allow the implementation of efficient algorithms for the analysis and comparison of β structures in proteins. A computer program for the representation and retrieval of bibliographic data and β sheet structures was implemented. Some examples for substructure search illustrate the usefulness of the program. Two graphic catalogues were compiled: one contains all β graphs of PDB proteins and the other all occurring different greek key descriptions.
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  • 67
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 345-364 
    ISSN: 0887-3585
    Keywords: protein structure ; crystallography ; errors ; φ,ψ distribution ; χ1 angles ; stereochemical parameters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Methods have been developed to assess the stereochemical quality of any protein structure both globally and locally using various criteria. Several parameters can be derived from the coordinates of a given structure. Global parameters include the distribution of φ,ψ and χ1 torsion angles, and hydrogen bond energies. There are clear correlations between these parameters and resolution; as the resolution improves, the distribution of the parameters becomes more clustered. These features show a broad distribution about ideal values derived from high-resolution structures. Some structures have tightly clustered distributions even at relatively low resolutions, while others show abnormal scatter though the data go to high resolution. Additional indicators of local irregularity include proline φ angles, peptide bond planarities, disulfide bond lengths, and their χ3 torsion angles. These stereochemical parameters have been used to generate measures of stereochemical quality which provide a simple guide as to the reliability of a structure, in addition to the most important measures, resolution and R-factor. The parameters used in this evaluation are not novel, and are easily calculated from structure coordinates. A program suite is currently being developed which will quickly check a given structure, highlighting unusual stereochemistry and possible errors.
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  • 68
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 382-399 
    ISSN: 0887-3585
    Keywords: protein secondary structure ; amino acid sequence ; distributions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The conditional probability, P(σ|x), is a statement of the probability that the value of σ will be found given the prior information that a value of x has been observed. Here σ represents any one of the secondary structure types, α, β, τ, and ρ for helix, sheet, turn, and random, respectively, and x represents a sequence attribute, including, but not limited to: (1) hydropathy; (2) hydrophobic moments assuming helix and sheet; (3) Richardson and Richardson helical N-cap and C-cap values; (4) Chou-Fasman conformational parameters for helix, Pα, for sheet, Pβ, and for turn, Pτ; and (5) Garnier, Osguthorpe, and Robson (GOR) information values for helix, Iα, for sheet, Iβ, for turn, I,τ, and for random structure, Iρ.Plots of P (σ|x) vs. x are demonstrated to provide information about the correlation between structure and attribute, σ and x. The separations between different P (σ|x) vs. x curves indicate the capacity of a given attribute to discriminate between different secondary structural types and permit comparison of different attributes. P (α|x), P (β|x), P (τ|x) and P (ρ|x) vs. x plots show that the most useful attributes for discriminating helix are, in order: hydrophobic moment assuming helix 〉 Pα » N-cap 〉 C-cap ≈ Iα ≈ Iτ. The information value for turns, Iτ, was found to discriminate helix better than turns. Discrimination for sheet was found to be in the following order: Iβ » Pβ ≈ hydropathy 〉 Iρ ≈ hydrophobic moment assuming sheet.Three attributes, at their low values, were found to give significant discrimination for the absence of helix: Iα ≈ Pα ≈ hydrophobic moment assuming helix. Also, three other attributes were found to indicate the absence of sheet: Pβ » Iτ ≈ hydropathy. Indications of the absence of σ could be as useful for some applications as the indication of the presence of σ.
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  • 69
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 26-37 
    ISSN: 0887-3585
    Keywords: norcamphor ; P450CIA1 ; substrate specificity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: While cytochrome P-450cam, catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) simulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the home iron and the substrate of about 1.0 Å; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphor-bound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex. © 1992 Wiley-Liss, Inc.
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  • 70
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 57-69 
    ISSN: 0887-3585
    Keywords: protein conformation ; aromatic contribution ; disulfide contribution ; CD spectra of the main secondary structural elements in proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure β-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:-669-679, 1991), was improved by the addition of proteins with high β-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (α-helix, antiparallel β-pleated sheet, β-turns, and unordered conformation), as well as a curve attributed to the “aromatic contribution” in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the α-helical structure, but upon analysis yielded a considerable amount of β-sheet in agreement with the X-ray structure. © 1992 Wiley-Liss, Inc.
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  • 71
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 120-131 
    ISSN: 0887-3585
    Keywords: protein folding ; α-helix ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A computational study of the role of all ionizable groups of the C-peptide in its helix-coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix-coil equilibrium of the C-peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix-coil transition due to the side chains of the C-peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix-coil transition of the C-peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed. © 1992 Wiley-Liss, Inc.
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  • 72
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 152-157 
    ISSN: 0887-3585
    Keywords: Staphylococcus aureus ; bacterial toxins ; structure-function ; protein structure ; crystallography ; pyrogenic toxins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 Å, respectively. The triclinic crystals have unit cell parameters a = 38.5 Å, b = 43.7 Å, c = 36.9 Å, and interaxial angles α = 99.9°, β = 95.8°, and γ = 98.5°. The tetragonal crystals are of space group P4122 with unit cell parameters a = 43.4 Å and c = 278.0 Å. © 1992 Wiley-Liss, Inc.
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  • 73
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 162-173 
    ISSN: 0887-3585
    Keywords: protein folding ; folding intermediates ; time resolved fluorescence ; nonradiative excitation energy transfer ; BPTI ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of BPTI and reduced BPTI in concentrated guanidinium HCI (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Inter probe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its ε-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states.The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 ± 2 Å) in the native state. Upon unfolding by reduction with DTT or β-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced averageinterprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted β sheet element (residues 18-35), increased upon unfolding. At -30°C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 ±20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 ± 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation.Thus local structures seem to exist in reduced denatured BPTI even underdenaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded folded proteins and for search for folding initiation sites (FISs) and early folding intermediates. © 1992 Wiley-Liss, Inc.
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  • 74
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 206-222 
    ISSN: 0887-3585
    Keywords: molecular docking ; Monte Carlo ; simulated annealing ; rational drug-design ; dihydrofolate reductase ; proteinase inhibitors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure. Our method is based on a Monte Carlo (MC) algorithm applied tothe translational and rotational degrees of freedom of the probe fragment. Starting from a randomly generated initial configuration, favorable bindingmodes are generated using a two-step process. An MC run is first prformed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the targetsurface. This has the effect of making buried probes move toward the targetsurface and also allows enhanced sampling of deep pockets. In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing). We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favourable docking modes in a specified region around the target. We test this method using two inhibitor-receptor systems: Streptomyces griseus Proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E. Coli in complex with methotrexate. The method could consistently reproduce the complex found in thecrystal structure searching from random initial positions in cubes ranging from 25 Å to 50 Å about the binding site. In the case of SGPB, we were also successful in docking to the native structure. In addition, we were successful in docking small probes in a search that included the entire protein surface. © 1992 Wiley-Liss, Inc.
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  • 75
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 272-272 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 76
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 258-271 
    ISSN: 0887-3585
    Keywords: protein folding ; protein modeling ; knowledge-based prediction ; molecular force field ; statistical mechanics ; globins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base andthe associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. Withone exception we find that for all globin sequences one of the known globinfolds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structurlresearch and future development of our approach. © 1992 Wiley-Liss, Inc.
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  • 77
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    Proteins: Structure, Function, and Genetics 14 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 78
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    Proteins: Structure, Function, and Genetics 13 (1992), S. 364-368 
    ISSN: 0887-3585
    Keywords: pancreatic spasmolytic polypeptide ; porcine pancreas ; hanging drop vapor diffusion method ; crystallization ; circular dichroism analysis ; porcine insulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X-ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I212121 with unit cell parameters a = 54.38 Å, b = 72.29 Å, and c = 180.85 Å. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 Å.The far-UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% α-helix, between 10 and 20% antiparallel β-strand, 10% parallel β-strand, 15% turn, and 25 to 40% of other structures. © 1992 Wiley-Liss, Inc.
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  • 79
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 1-9 
    ISSN: 0887-3585
    Keywords: protein engineering ; cassette mutagenesis ; peptide hormone ; molecular modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: By inserting appropriate peptide ligands into surface loops on globular proteins, we expect to develop probes for the location, accessibility, and steric and electrostatic environment of these ligand-binding sites on their membrane-bound receptors. Three residues in a loop on the surface of E. coli alkaline phosphatase were substituted by an 18-residue peptide containing the receptor-binding segment of somatostatin-14 without significantly affecting the catalytic properties of the enzyme. This hybrid protein was then used to investigate the ligand-binding site of somatostatin receptors. Tryptic cleavage of the hybrid protein within the inserted sequence, and binding of the hybrid protein to antisomatostatin antibodies demonstrated the surface accessibility of the guest peptide. Both the wild-type enzyme and the hormone-enzyme hybrid displaced 125I-labeled somatostatin from rat brain membrane receptors only at high concentrations. How-ever, chemical cationization of the hybrid protein, which again did not disturb the phosphatase activity, enhanced its receptor-binding potency to a level only 23 times lower than that of somatostatin itself and 280 times higher than that of the cationized wild-type protein. This alkaline phosphatase/somatostatin hybrid protein appears, therefore, to be a suitable starting point for the development of probes for the steric and electrostatic environment of the ligand-binding site of somatostatin receptors. © 1992 Wiley-Liss, Inc.
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  • 80
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 168-177 
    ISSN: 0887-3585
    Keywords: protein structure ; helix-turn-helix motif ; Tet represser ; mutagenesis ; structure predictions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Tn10derived Tet represser contains an amino acid segment with high homology to the α-helix-turn-α-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first α-helix without loss of activity. The last position of the first α-helix, the second position in the turn, and the fourth position in the second α-helix require mostly hydrophobic residues. The proposed C-terminus of the first α-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented. © 1992 Wiley-Liss, Inc.
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  • 81
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 191-201 
    ISSN: 0887-3585
    Keywords: common protein fold ; protein architecture ; close packing ; p-sheet twist ; galactose oxidase ; influenza virus neuraminidase ; methylamine dehydrogenase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Twisted β-sheets, packed face to face, may be arranged in circular formation like blades of a propeller or turbine. This β-pro-peller fold has been found in three proteins: that in neuraminidase consists of six β-sheets while those in methylamine dehydrogenase and galactose oxidase are composed of seven β-sheets. A model for multisheet packing in the β-propeller fold is proposed. This model gives both geometrical parameters of the β-propellers composed of different numbers of sheets and patterns of residue packing at their sheet-to-sheet interfaces. All the known β-propeller structures have been analyzed, and the observed geometries and residue packing are found to be in good agreement with those predicted by models. It is shown that unusual seven-fold symmetry is preferable to six- or eight-fold symmetry for propeller-like multisheet assembly. According to the model, a six β-sheet propeller has to have predominantly small residues in the β-strands closed to its sixfold axis, but no strong sequence constraints are necessary for a seven-fold β-propeller. © 1992 Wiley-Liss, Inc.
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  • 82
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 178-190 
    ISSN: 0887-3585
    Keywords: flavoenzymes ; monooxygenase ; FAD ; reduced flavin ; flavin planarity ; Pseudomonas fluorescens ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonasm fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy.X-ray data to 2.3 Å were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 Å structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules.The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 Å. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2° This value should be compared with observed values of 10° for the oxidized enzyme-substrate complex and 19° for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found. © 1992 Wiley-Liss, Inc.
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  • 83
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 202-212 
    ISSN: 0887-3585
    Keywords: semisynthesis ; protein structure ; ligand binding ; amino acid substitution ; solid-phase peptide synthesis ; hydrophobicity ; reduction potentials ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R.E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a “control” analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket. © 1992 Wiley-Liss, Inc.
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  • 84
    ISSN: 0887-3585
    Keywords: subdomain ; kinetics ; unfolding ; stabilization ; autolysis ; protein engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue β-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the β-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the β-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the inter action between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis. © 1992 Wiley-Liss, Inc.
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  • 85
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 249-264 
    ISSN: 0887-3585
    Keywords: fuzzy clustering ; molecular dynamics ; simulations ; nonequilibrium ; protein conformation ; parathyroid hormone (PTH) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We propose fuzzy clustering as a method to analyze molecular dynamics (MD) trajectories, especially of proteins and polypeptides. A fuzzy cluster analysis locates classes of similar three-dimensional conformations explored during a molecular dynamics simulation. The method can be readily applied to results from both equilibrium and nonequilibrium simulations, with clustering on either global or local structural parameters. The potential of this technique is illustrated by results from fuzzy cluster analyses of trajectories from MD simulations of various fragments of human parathyroid hormone (PTH). For large molecules, it is more efficient to analyze the clustering of root-mean-square distances between conformations comprising the trajectory. We found that the results of the clustering analysis were unambiguous, in terms of the optimal number of clusters of conformations, for the majority of the trajectories examined. The conformation closest to the cluster center can be chosen as being representative of the class of structures making up the cluster, and can be further analyzed, for example, in terms of its secondary structure. The CPU time used by the cluster analysis was negligible compared to the MD simulation time. © 1992 Wiley-Liss, Inc.
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  • 86
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 237-248 
    ISSN: 0887-3585
    Keywords: protein folding ; denatured states ; hydrogen exchange ; NMR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30°C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 105-107 times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35°C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide crosslink (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69°C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme. © 1992 Wiley-Liss, Inc.
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  • 87
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 265-276 
    ISSN: 0887-3585
    Keywords: representative PDB structures ; sequence clustering ; significance of sequence similarity ; classification of protein structures ; amino acid composition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of α-and β-structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of α-and β-structures even when sequence similarity is relatively low.An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjectice view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics. © 1992 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
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  • 88
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 277-287 
    ISSN: 0887-3585
    Keywords: α-helix ; lysine residues ; disaccharide units ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (I→4)-O-(α-L-idopyranosyluronic acid 2-sulfate)-(l→4)-(2-deoxy-2-sulfamino-2-D-glucopyra-nosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 Å resolution with R = 0.237. Each monomer of BPF4 contains an α-helix lying across 3 strands of antiparallel β-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the α-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 89
    ISSN: 0887-3585
    Keywords: amino acid-derived cofactor ; crystal structure ; methylamine dehydrogenase ; molecular replacement ; oxidoreductase ; Paracoccus denitrificans ; pyrroloquinoline quinone ; quinoprotein ; tryptophan tryptophylquinone ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans (PD-MADH) has been determined at 2.8 Å resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an “X-ray” sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded β-segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 β-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two co-valently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 90
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 300-308 
    ISSN: 0887-3585
    Keywords: polyethylene glycol-400 ; phosphoglucomutase crystals ; salt solutions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol-400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long-term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG-induced increase in the “solubility” of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute-induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyeth-yiene glycol and β-octylglucoside on amorphous, salt-induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility-based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described. © 1992 Wiley-Liss, Inc.
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  • 91
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    Proteins: Structure, Function, and Genetics 14 (1992), S. 309-323 
    ISSN: 0887-3585
    Keywords: protein structure comparison ; sequence alignment ; structure alignment ; dynamic programming ; dehydrogenase fold ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root The algorithm encoded in STAMP (Structural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases.In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij′ provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij′ values and thus provide a reproducible resource for studies of residue conservation within structural motifs. © 1992 Wiley-Liss, Inc.
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  • 92
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    Proteins: Structure, Function, and Genetics 12 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 93
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 1-9 
    ISSN: 0887-3585
    Keywords: hematopoiesis ; colony-stimulating factors ; structure-function relationships ; GM-CSF ; IL-3 ; helical proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The hematopoietic growth factors are a family of glycoproteins involved in the production of blood cells from their bone marrow precursors and in the activation of mature blood cells. Much has been learned about the structural features of these molecules responsible for their characteristic biological activities. Most studies have been based upon mutagenesis strategies of intact polypeptides and on epitope mapping of informative monoclonal antibodies to the growth factors. A more limited amount of physical data is available. This review will summarize these findings, highlight the growing body of evidence suggesting that many of these proteins share common evolutionary origins and structural elements, and hopefully point to the directions being taken for further investigations of these scientifically informative and clinically useful group of proteins.
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  • 94
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 49-62 
    ISSN: 0887-3585
    Keywords: β-helix ; circular dichroism ; tryptophan ; phenylalanine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In order to resolve whether gramicidin A channels are formed by right- or left-handed β-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therfore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed β6.3-helical dimers.
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  • 95
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 87-90 
    ISSN: 0887-3585
    Keywords: crystallization ; X-ray diffraction ; immunoaffinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The class I major histocompatibility (MHC) antigen HLA-B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG-2. Detergent-soluble HLA-B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water-soluble extracellular fragments were obtained in hanging drops by the vapor-diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 Å, b = 71.0 Å, c = 83.7 Å, α = 79.4°, β = 88.5°, γ = 89.9°, and diffract beyond 2.5 Å resolution.
    Additional Material: 3 Ill.
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  • 96
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 10-23 
    ISSN: 0887-3585
    Keywords: crystallography ; molecular isomorphous replacement ; molecular dynamics ; refinement ; interleukin ; site-directed mutagenesis ; receptor-binding surface ; epitope ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The molecular structure of interleukin-1β, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 Å resolution and refined to a crystallographic R-factor of 0.19. The frame-work of this molecule consists of 12 antiparallel β-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a β-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the β-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.
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  • 97
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 42-48 
    ISSN: 0887-3585
    Keywords: calcium ; zinc ; tryptophan fluorescence ; denaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 ∼60 μM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.
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  • 98
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 100-100 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 99
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    Proteins: Structure, Function, and Genetics 12 (1992), S. 24-30 
    ISSN: 0887-3585
    Keywords: interleukin-2 ; protein crystallography ; glycoprotein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6222 or its enantiomorph. The crystals diffract to 2.8 Å and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5-5.6 in the monoclinic space group P21, and less frequently in the orthorhombic space group P212121 from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3121 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals.
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  • 100
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    Proteins: Structure, Function, and Genetics 12 (1992) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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