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  • 1985-1989  (3,485)
  • 1975-1979  (1,448)
  • Biochemistry and Biotechnology  (3,982)
  • Ultrastructure  (951)
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Year
  • 1
    ISSN: 1432-0428
    Keywords: Islet amyloid polypeptide ; Pancreatic islets ; B cells ; Ultrastructure ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Islet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized a rabbit antiserum against a synthetic peptide corresponding to positions 20–29 of islet amyloid polypeptide, a sequence without any amino-acid identity with calcitonin gene-related peptide. By applying the immunogold technique at the ultrastructural level, it was shown that both insulin and islet amyloid polypeptide immunoreactivity occurs in the central granular core of the human B cell secretory granules, while the A cells remain unlabelled. The demonstration that islet amyloid polypeptide is a granular protein of the B cells may indicate that it is released together with insulin. Further studies are necessary to evaluate the functional role of islet amyloid polypeptide.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Development genes and evolution 198 (1989), S. 92-102 
    ISSN: 1432-041X
    Keywords: Vitellogenesis ; Xenopus oocyte ; Yolk-platelet membrane ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Throughout vitellogenesis, large yolk platelets are in close contact with smaller nascent yolk organelles. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets was not observed. On freezeetching replicas, yolk-platelet membranes present fracture faces with intramembranous particles (IMP) of various sizes and a heterogeneous distribution of approximately 200–600 IMP/μm2 at the E face, and 1200–2100 IMP/μm2 at the P face. Again, this presentation of the membrane exhibits neither anastomoses to the endoplasmic reticulum, nor caveolae that exclude the uptake of yolk-containing vesicles into these yolk organelles. Proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.
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  • 3
    ISSN: 1432-0568
    Keywords: Monkey ; Ultrastructure ; Pinealocytes ; Axon terminals ; Synapses
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The present study described the normal ultrastructure of the monkey pineal gland. The gland was composed of the principal pinealocytes, intramural neurons and glial cells. The nucleus of the pinealocytes was deeply infolded with evenly distributed chromatin materials. The abundant cytoplasm was rich in organelles including the well-developed Golgi apparatuses, multivesicular bodies, dense-cored vesicles and widely scattered free and polyribosomes. A variety of axon terminals was observed and the majority of them contained pleomorphic agranular vesicles with a few large dense-cored vesicles. A few terminals showed flattened vesicles or small dense cored vesicles. Some of the axon terminals formed synaptic contacts with the cell bodies of pinealocytes. These synapses were mainly concentrated in the posterior third of the gland. The occasional intramural neurons observed were postsynaptic to axon terminals containing round agranular vesicles. The sources of the nerve fibres and terminals forming synaptic junctions with pinealocytes and intramural neurons were discussed.
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  • 4
    ISSN: 1432-0568
    Keywords: Sympathetic ganglion ; Binucleate cells ; Ultrastructure ; Feulgen staining ; Computerized image analysis ; DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The relative proportion, ultrastructure and DNA-content of the binucleate cells in the celiac superior mesenteric ganglion of the guinea pig was studied using light and electron microscopy as well as computerized image analysis of Feulgen stained cells. The number of mono — versus binucleate cells was found to vary with stage of development with about 40% of the cells being binucleate in adult animals and 50% in late prenatal stage. No difference in ultrastructure was observed between the nuclei of the two cell types. The binucleate cells contain twice the amount of DNA found in the mononucleate cells.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 180 (1989), S. 175-178 
    ISSN: 1432-0568
    Keywords: Paratympanic organ ; Reciprocal synapses ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The innervation pattern of the paratympanic organ was studied by TEM. The paratympanic organ is a small tapering vesicle, typical of birds, situated in the medial wall of the tympanic cavity; it contains hair cells which are similar to type II receptors of the acoustic-lateral system; these cells are characterised by synapses which are not only afferent and efferent, as previously described, but also reciprocal with efferent fibers. Our observation revealed some efferent nerve fibers which form a relationship with hair cells containing synaptic bodies situated next to the plasma membrane and near the fibers themselves. Since synaptic bodies are commonly considered to be the site where the transmission of the impulse from the receptor to the nerve fiber takes place, our pictures suggest that the efferent fibers and hair cells may be either presynaptic or postsynaptic with respect to each other in the paratympanic organ. The hypothesis is formulated that reciprocal synapses allow interaction between hair cells, thus determining an increase in the contrast of information sent by the paratympanic organ to the CNS.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 179 (1989), S. 591-604 
    ISSN: 1432-0568
    Keywords: Fetus ; Membranes ; Placenta ; Green monkey ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary This study examined developmental changes in fetal membranes and placenta of Cercopithecus aethiops from a Carnegie developmental stage 12 embryo to nearterm fetuses. Ultrastructurally, yolk sac cells (endoderm and mesothelium) were similar to comparable stages in other primates. Endodermal cells had few apical microvilli, abundant rough-endoplasmic reticulum, electron dense mitochondria and dense bodies. In contrast, mesothelial cells were squamous with numerous microvilli, small mitochondria and a few short strands of rough endoplasmic reticulum. Amnion cells early in gestation were squamous with few microvilli, large glycogen deposits and poorly developed cytoplasmic components. Tight junctions and desmosomes held adjacent cells together. The basal surface was smooth and the basal lamina was distinct. As development proceeded the amniotic cells became cuboidal and possessed numerous microvilli. Cytoplasmic organelles were better developed and glycogen deposits increased by mid-gestation. A thick layer of microfibrils and collagen fibers was prominent below the basal lamina. Near-term, the glycogen had virtually disappeared and the amount of lipid droplets increased. Basal infoldings and podocytic processes and the extracellular matrix had increased. The smooth chorion consisted of pseudostratified columnar cells. Cells had short microvilli, numerous granules and vesicles of variable size and electron density in early gestation. With increasing age, amounts of granules and vesicles decreased, as the endoplasmic reticulum became prominent. The chorionic trophoblast was a continuous layer in mid-pregnancy and its cells had well-developed organelles and inclusions. Late in gestation, the trophoblastic layer became discontinuous and wide intercellular spaces and channels were present. In the placenta, the trophoblastic elements showed features characteristic of primate placenta.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 179 (1989), S. 435-442 
    ISSN: 1432-0568
    Keywords: Ultrastructure ; In vitro fertilization ; Bovine ; Ova ; Cortical granules
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Heifers were superovulated by PMSG or FSH, and oestrus was induced by prostaglandin. One group of animals was ovariectomized 19–26 h after the LH peak, the content of preovulatory follicles aspirated, and the oocytes processed for in vitro fertilization. Another group was inseminated and ova were collected from the oviducts for study of in vivo fertilization. All ova were examined ultrastructurally. The developmental rate following in vitro fertilization was delayed compared to fertilization in vivo. A high proportion of the in vitro fertilized ova showed polyspermic penetration of the zona pellucida, and supernumerary spermatozoa were found in the ooplasm of some ova. In vivo fertilization was associated with release and subsequent dispersal of the cortical granule content in the perivitelline space. In contrast to this the released granule content of the in vitro fertilized ova remained undispersed close to the oolemma. This feature may account for the high incidence of polyspermic penetration of the zona pellucida. In addition, the study provided an ultrastructural visualization of the initial contact between the equatorial segment of the spermatozoon and the microvilli of the oocyte, and the subsequent internalization of the sperm head.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 179 (1989), S. 497-501 
    ISSN: 1432-0568
    Keywords: Parotid gland ; Ultrastructure ; Amylase ; Secretion ; Isoproterenol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The effects of continuous light on ultrastructural organization and sympathetic secretory responses of the rat parotid gland are reported. After 50 days of continuous light exposure, the fine structure of the parotid gland exhibited features of enhanced secretory activity as judged by the striking development of rough endoplasmic reticulum and Golgi complexes, the depletion of secretory granules and the increased turnover of secretory cells. The secretory responses of parotid gland to isoproterenol revealed that continuous light induced a 30% increase in amylase release. This secretory hyperactivity appears to be related to a postsynaptic supersensitivity of sympathetic fibers of the autonomic nervous system.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 180 (1989), S. 103-108 
    ISSN: 1432-0568
    Keywords: Ultrastructure ; Gut ; Endocrine cells ; Testudo graeca ; Chelonia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The digestive tract of Testudo graeca (Chelonia) was investigated by means of electron microscopy using both conventional and immunocytochemical techniques. EC-, L-, D-, G-, B-, N- and EC-L-cells were detected. These cells share several common ultrastructural characteristics with the endocrine cells of mammals (i.e. clear cytoplasm, prominent Golgi apparatus, secretory granules etc.). EC and D1 cells have so far not been described in the esophagus of any animal species; in the present study these cells have been observed in the esophagus of T. graeca. Of special interest was the presence of B-cells in the intestine, suggesting that the migration of B-cells from the gut to the pancreas to constitute pancreatic islets is not concluded in T. graeca. The present study demonstrates that the gut endocrine system of T. graeca is a complex structure containing a large variety of endocrine cell types similar in morphology to those found in higher vertebrates.
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  • 10
    ISSN: 1432-0568
    Keywords: Cecum ; Germ-free rat ; Microflora inoculation ; Morphometry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The structural changes of the cecal wall in germfree rats were observed at regular intervals after the inoculation of fecal microflora from conventional rats. Quantitative light microscopy showed that most of the elements in the cecal wall increased at 12 or 24 h and reached peak values at 4 days after inoculation. On the 7th day, they decreased approximately to the values for conventional rats. The crypts were bent or widely open till 24 h but were not after the 4th day. Hyperplasia of the crypt epithelial cells including mucous-type cells was observed following microbial inoculation. Electron microscopy revealed that most of the epithelial cells lining the mucosa were typical columnar cells. Desquamation of the epithelial cells and contraction of the muscle fibers were often seen on 4th day. The mucous-type cells were divided into two types, goblet and non-goblet mucous-type cells. Reduction of cecal volume after microbial inoculation may be mainly caused by muscle contraction in the early period and hyperplasia and desquamation of the epithelial cells may suggest their role as the first and non-specific defense line prior to operation of the specific immune system.
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 77 (1989), S. 258-266 
    ISSN: 1432-0533
    Keywords: Neurofibrillary tangles ; Alzheimer's disease ; Pick bodies ; Immunohistochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We have studied the immunohistochemical reactivity and ultrastructure of both neurofibrillary tangles (NFTs) occurring with severe neurofibrillary diseases, and Pick bodies (PBs) associated with Pick's disease. The NFTs and PBs did not react immunohistochemically with the anti-nonphosphorylated neurofilament monoclonal antibody irrespective of whether they were pretreated with alkaline phosphatase. In granular neurons of the dentate fascia of Ammon's horn in cases of dementia of the Alzheimer type (DAT), NFTs either resembled PB-like inclusion bodies (Horoupian's inclusion bodies) in form, or had a perinuclear structure. Immunohistochemically and ultrastructurally, the NFTs in the dentate fascia in cases of DAT, including Horoupian's inclusion bodies, were similar to the NFTs in the pyramidal neurons of Ammon's horn, which are found most frequently in association with severe neurofibrillary diseases. Under a light microscope, Horoupian's inclusion bodies and PBs could not be differentiated and appeared to be argyrophilic round cytoplasmic inclusions in granular neurons of the dentate fascia. There were, however, ultrastructural differences. Horoupian's inclusion bodies consisted of bundles made up of straight tubules (STs), each about 15 nm in diameter. These bundles were intermixed with a few paired helical filaments which occurred at intervals of about 80 nm. On the other hand, PBs were composed of randomly distributed 15-nm-wide STs, intermixed with a very few fibrillary structures. These fibrils had a periodicity of about 160 nm, and ranged in width from about 15 nm to 30 nm. Horoupian's inclusion bodies associated with DAT and PBs associated with Pick's disease are different in this neuropathological aspect. The NFTs, including Horoupian's inclusion bodies in the dentate fascia in cases of DAT, are considered to be a manifestation of neurofibrillary degeneration.
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 77 (1989), S. 654-658 
    ISSN: 1432-0533
    Keywords: Melanotic neuroectodermal tumor ; Immunohistochemistry ; Ultrastructure ; Pineal origin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A case of a melanotic neuroectodermal tumor arising from pineal region of a 4-year-old girl is presented. The tumor had spread diffusely to the meninges, consistent with malignant behavior. Histologically, the tumor consisted primarily of epithelial elements arranged in tubules, cords and nests separated by fibrous vascular tissue in addition to a small neuroblastomatous focus. Melanin pigment was frequently observed in the epithelial tumor cells, and melanin-laden macrophages were also often observed. No teratoid elements were found. Immunohistochemically, tumor cells were positive for neuron-specific enolase but were nonreactive for S-100 protein, epithelial membrane antigen, glial fibrillary acidic protein, vimentin, α-fetoprotein and human chorionic gonadotrophin. Ultrastructurally, the epithelial nature of the tumor cells could be easily demonstrated. In addition, melanosomes in various stages in maturation were observed, indicating melanogenesis of the tumor. On the basis of the tumor location and the histological similarities previously observed for the fetal pineal body, it is very likely that this melanotic epithelial tumor could have originated from the fetal pineal gland.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 79 (1989), S. 86-93 
    ISSN: 1432-0533
    Keywords: Primary malignant CNS lymphoma ; Ultrastructure ; Intracytoplasmic tubuloreticular, membranous structures ; Intranuclear inclusions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Ultrastructural studies of 17 primary malignant CNS lymphomas revealed 6 tumors with abnormal intracytoplasmic and/or intranuclear membranous structures, most frequently, associated with the endoplasmic reticulum or perinuclear envelope. In most cases, tubuloreticular inclusions and paired cisternae were present. Less frequent were accumulation of mictotubules, concentric lamellar bodies, and rod-like or paracrystalline intranuclear inclusions. The specificity and significance of these membranous structures remain questionable because of their frequent occurrence in a variety of normal and pathological conditions. Some of these changes may be considered as cellular reactions to viral infections, others may indicate cellular activity or degeneration.
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  • 14
    ISSN: 1432-0533
    Keywords: 2,4-Dithiobiuret ; Thioimidodicarbonic diamide ; Motor endplate ; Neuromuscular junction ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary 2,4-Dithiobiuret was given i.p. to rats for 4 days at a daily dosage of 1 mg/kg and the development of the lesion associated with neuromuscular dysfunction studied in hindlimb lumbrical muscles. The first morphological indication of neurointoxication was the appearance in some motor endplates of masses of branching tubular smooth endoplasmic reticulum (SER) on day 2 which correlated with the initial functional disturbances. By the 3rd day, most motor endplates were distended by accumulations of densecored, lucent and synaptic vesicles, abnormally swollen mitochondria, intermediate filaments and branching, tubular SER. Evidence of collateral axonal sprouting was seen first at this time. On days 4 and 5, many motor endplates were markedly enlarged and showed axoplasmic organelle congestion. A significant increase in synaptic vesicle size was noted at these times in some terminals. Interposition of Schwann cell processes between the pre- and postsynaptic membranes and terminal retraction was now evident. Some intramuscular nerves showed hydropic Schwann cell cytoplasm with separation of the outermost myelin lamellae, mitochondrial swelling and adaxonal vacuoles as early as the 1st day. Proliferation and segregation of SER around central cores of neurofilaments was seen in myelinated nerve fibres and preterminals on the 3rd day. At this and later times accumulations of SER and swollen mitochondria were found at sites of axonal varicosities and at the paranodal constrictions at nodes of Ranvier. These ultrastructural data are discussed with regard to reduced terminal Ca2+ content (demonstrated by oxalate-pyroantimonate cytochemistry) and compared with the sequelae of botulinum intoxication.
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  • 15
    ISSN: 1432-0533
    Keywords: Gangliocytoma ; Ganglioglioma ; Ultrastructure ; Immunohistochemistry ; Neuroendocrine markers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We studied five cases of central nervous system neuronal tumor, one gangliocytoma and four gangliogliomas, both ultrastructurally and immuno-histochemically, using antibodies to neuroendocrine markers including tyrosine hydroxylase (TH), serotonin (5HT), somatostatin (SOM), met-enkephalin (MEK), leu-enkephalin (LEK), substance P (SP), gastrin, vasopressin, oxytocin, vasoactive intestinal polypeptide, adrenocorticotropic hormone and calcitonin. In all cases, the presence of dense-core vesicles (60–250 nm) in the neuronal elements was the characteristic ultrastructural finding. Synapses were observed in two cases. Immunohistochemically, variable numbers of neuronal cells showed positive staining for SOM in five cases, TH, MEK and LEK in three cases, and 5HT and SP in one case each. The others were negative. Positive immunoreactivity for multiple markers was shown in all cases. SOM, TH, 5HT and SP were present in the small- to medium-sized cells, while MEK and LEK were almost exclusively confined to the large cells. Our study clearly indicated that these tumors contained neuronal cells which were not homogeneous with regard to neuroendocrine markers.
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  • 16
    ISSN: 1432-0533
    Keywords: Neuronal inclusions ; Leigh disease ; Tropomyosin ; Actin ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A novel type of non-viral cytoplasmic inclusion is described, which was seen in virtually every neuron in the brain and spinal cord of a child with a presumed metabolic disorder whose clinical picture and CNS pathology were compatible with Leigh Syndrome. The ovoid to round inclusions were sharply demarcated, measuring up to 11 μm in diameter. They showed no distinctive staining with a battery of routine histological techniques. The ultrastructural features are unique, comprising non-membrane-bounded aggregates of randomly oriented plate-like structures with parallel linear densities depicting a periodicity of 11–16 nm. Immunocytochemical studies revealed strong staining with antisera to tropomyosin and weaker staining with antisera to actin. There was no reactivity with antibodies against neurofilaments, microtubules and their associated proteins, paired helical filaments, ubiquitin, vinculin or alpha-actinin. It is postulated that the metabolic disorder resulted in a neurodegenerative condition which manifested pathologically with lesions compatible with those of Leigh Syndrome. Associated with the condition was the discrete accumulation of cytoplasmic proteinaceous components, including tropomyosin, in the form of neuronal cytoplasmic inclusions possibly resulting from an alteration of the neuronal cytoskeleton.
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  • 17
    ISSN: 1432-0533
    Keywords: Cerebral hypoxia ; Cerebral ischemia ; Ultrastructure ; Neocortex ; Brain isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The acute cortical response to surgical brain isolation and subsequent extracorporal normoxic or 30 min hypoxic (PaO2=20 mm Hg) perfusions (hypoxic hypoxia with relative ischemia) was evaluated. Cerebral blood flow, arterial pH and CO2 were maintained constant during both perfusions; only the arterial oxygen content was changed. The isolated brain model used in this and previous investigations produces no qualitative ultrastructural changes in the neocortex following brain isolation and normoxic perfusion. However, the acute cortical structural response to 30 min of hypoxic hypoxia with relative ischemia demonstrated a number of important observations. Hypoxic hypoxia produced ultrastructural responses common to cerebral ischemia such as nuclear chromatin clumping, nucleolar condensation and cytoskeletal breakdown. Although neuronal abnormalities seen after 30 min of hypoxic hypoxia were similar to those acute neuronal changes observed following complete cerebral ischemia without recirculation, they differed three ways: (a) mitochondrial swelling and microvacuolation were observed in many cortical pyramidal neurons. (b) Glycogen particles within astroglial processes were observed even after a 30-min period of hypoxic hypoxia. (c) Perivascular astroglial swelling was minimal despite considerable perineuronal swelling. In contrast, incomplete cerebral ischemia produces mitochondrial changes similar to those in hypoxic hypoxia but also causes the depletion of tissue glycogen and perivascular glial swelling. Thus, hypoxic hypoxia with relative ischemia produces a unique acute ultrastructural response compared to either complete or incomplete cerebral ischemia.
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 79 (1989), S. 336-339 
    ISSN: 1432-0533
    Keywords: AIDS ; Cytomembranous inclusions ; Tubuloreticular inclusions ; Ultrastructure ; Peripheral nerve
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We report finding tubuloreticular inclusions (TRI) in the endothelial cells of endo- and epineurial vessels in the sural nerve of 11 patients with AIDS. Six patients had a painful peripheral neuropathy, one a non-painful sensory neuropathy, one an acute inflammatory demyelinating polyradiculoneuropathy and one a thalidomide-related neuropathy. Two patients had no clinical evidence of neuropathy. The TRI are not specific to one neuropathy and are unlikely to contribute to the pathogenesis of peripheral nerve syndromes in AIDS.
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  • 19
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 79 (1989), S. 168-175 
    ISSN: 1432-0533
    Keywords: Muscular ; Dystrophy ; Ovine ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The initial ultrastructural changes in skeletal myofibers in ovine muscular dystrophy (MD) consisted of focal degeneration of myofibrils and the formation of Z-disc abnormalities, including nemaline rods, in adjacent sarcomeres. Peripheral and central sarcoplasmic masses, which occurred initially in large diameter fibers, contained a mixture of normal organelles and abnormal tubular and fibrillar formations. Vesiculate sarcolemmal nuclei with prominent nucleoli accumulated in central and subsarcolemmal locations in small clusters and short rows. Deformed individual nuclei were sometimes present within nuclear rows. Loss of the myofibrillar mass, increased density of small spherical nuclei, collections of fibrillar and tubular arrays, excessive folding of the sarcolemma and greatly reduced fiber diameter were seen in the end stage of the dystrophic process. Resting satellite cells were present at all stages of lesion development. The morphological progression of the lesions suggested an inherited inability to effectively replace lost myofibrils with ultimate exhaustion of the capacity for repair followed by pathological fiber atrophy.
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  • 20
    ISSN: 1432-072X
    Keywords: Thiothrix sp. ; Beggiatoa sp. ; Sulfideoxidizing ; Polyunsaturated ; Fatty acids ; Inclusions ; Sheath ; Southern California ; Ultrastructure ; Sulfur
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Microscopic examination of the whitish mat that covered the substrata around subtidal hydrothermal vents at White Point in southern California revealed a “Thiothrix-like” bacterium containing sulfur inclusions as the dominant filamentous form in this microbial community. The matlike appearance developed as a result of the closely-packed manner inwhich the basal ends of the filaments were anchored to the substrate. The dominant phospholipid fatty acids of these filaments (16:0, 16:1w7c, 18:0, 18:1w7c) were similar to those recovered from a sample of Beggiatoa isolated from a spring in Florida. Filaments from both sources contained small quantities of C18 and C20 polyunsaturated fatty acids, as well. A larger but less abundant sheathless, filamentous form, which also contained sulfur inclusions and displayed a cell wall structure similar to a previously described Thioploca strain, also colonized the substrata around the subtidal mat. The preservation methods used in the preparation of thin-sections of the subtidal mat material were found to be inadequate for defining some key cellular structures of the large filaments. Nevertheless, the results demonstrate that the filamentous bacteria comprising the microbial mat in the vicinity of the subtidal vents exhibit some of the features of the free-living filamentous microorganisms found in deep-water hydrothermal areas.
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  • 21
    ISSN: 1432-072X
    Keywords: Gallionella ferruginea ; Thiobacillus ferrooxidans ; Iron bacteria ; Chemolithoautotrophy ; Ultrastructure ; Freeze-etching ; Cell wall organization ; Intracytoplasmic membranes ; Carboxysomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly-β-hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.
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  • 22
    ISSN: 1432-072X
    Keywords: Methanobacterium formicicum ; Formate dehydrogenase ; F420-hydrogenase ; Immunogold ; Ultrastructure ; Methanogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ultrastructural locations of the coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F420-dependent formate dehydrogenase activity or F420-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.
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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    Virchows Archiv 414 (1989), S. 459-464 
    ISSN: 1432-2307
    Keywords: Sebaceous carcinoma ; Parotid gland ; Salivary gland ; Ultrastructure ; Immunohistochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Sebaceous carcinoma of salivary gland origin is extremely rare and, because of its rarity, the clinicopathological characteristics and the histogenesis are not fully understood. We present a case of sebaceous carcinoma of the parotid gland which brings the total number of reported cases to 22. The tumor showed epithelial cell nests which were mainly composed of sebaceous cells with marked cellular atypia. In most of the nests, glandular spaces lined by ductal epithelium were present. Scattered mucous cells and flattened eosinophilic cells at the periphery of the nests were also seen. Ultrastructural and immunohistochemical observations of the tumour revealed coexistence of sebaceous and glandular differentiations in some tumour cells. Tumour cells with lipid granules often participated in the formation of glandular structures or exhibited intracytoplasmic lumina, and immunohistochemical localization of lactoferrin and secretory component, the functional markers of ductal epithelium of salivary gland, was demonstrated not only in duct-forming tumour cells but also in many sebaceous tumour cells. It seems likely that sebaceous carcinoma originates from pluripotential duct cells which can differentiate into sebaceous, ductal and mucous cells.
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  • 24
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    Virchows Archiv 415 (1989), S. 145-149 
    ISSN: 1432-2307
    Keywords: Paracrystalline inclusion ; Microtubule ; Ciliogenesis ; Gastric ciliated cell ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Unusual electron-dense paracrystalline inclusions were found in metaplastic ciliated cells in the stomachs of three Japanese male patients with gastric carcinoma. These patients had not been given antitumour drugs before surgery and ethrane (enflurane) was used as the anaesthetic. Ciliated cells in the gastric mucosa are found not infrequently in the pyloric glands in association with intestinal metaplasia in elderly Japanese patients. Paracrystalline inclusions were found only in the ciliated cells and never in any other types of gastric mucosal cell. These inclusions were located in the apical portion of the ciliated cells in intimate association with the basal bodies. They consisted of twisted strings about 27 nm wide with a regularly repeated spacing of about 30 nm. On highly magnified electron micrographs, granules about 4 nm in diameter were detected. These paracrystalline inclusions have never been reported previously, although their location in ciliated cells and their morphological characteristics suggest an intimate relationship with the ciliogenesis of metaplastic ciliated cells in the human stomach.
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  • 25
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    Virchows Archiv 414 (1989), S. 113-119 
    ISSN: 1432-2307
    Keywords: Gastric mucosa ; Intestinal metaplasia ; Ciliated cell ; Ciliated metaplasia ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Intestinal metaplasia of the gastric mucosa occurs commonly in aged Japanese patients and has been discussed in relation to the high incidence of gastric cancer in Japanese. Ciliated cells in the gastric mucosa have frequently been found in association with intestinal metaplasia in the pyloric gland and rarely in the cardiac gland in many Japanese patients, and exceptionally in one Chinese and in one Swedish patient. Electron microscopic examination of 12 Japanese patients has revealed that these structures are not metaplastic stereocilia, but true cilia. Ciliated cells have been found in the basal part of the gastric glands and never in the surface epithelium. The fine structure of the gastric cilia was almost the same as that of normal respiratory cilia. However, in the gastric cilia, most dynein arms were inconspicuous even after tannic acid fixation, indicating that ciliary beating of the gastric cilia is problematic. Abnormal cilia and basal bodies also were found. Ciliated cells have always occurred in association with intestinal metaplasia, therefore this phenomenon might be a type of metaplasia and is named “ciliated metaplasia” of the gastric mucosa.
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  • 26
    ISSN: 1432-2307
    Keywords: Malignant fibrous histiocytoma ; Ultrastructure ; Enzyme histochemistry ; Immunohistochemistry ; “Fibrohistiocytoid cell”
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Ultrastructural, enzyme histochemical and immunohistochemical studies were performed on tissue obtained from eight cases of malignant fibrous histiocytoma (MFH) and five cases of sacral decubitus ulcer. The MFH was composed of two major tumour cell types: fibroblast-like and histiocyte-like cells. Both cell types demonstrated abundant branching, fragmented rough endoplasmic reticulum (rER), many free ribosomes, occasional small mitochondria, an oval, elliptical or irregularly shaped nucleus with one or two prominent nucleoli and often a few dense bodies. However, pseudopodial projections, multivesicular bodies and phagosomes, common histiocyte organelles, were not seen. With little difference between cases or selection sites, the MFH cells reacted to acid phosphatase (AcP) and α-naphtyl butyrate esterase (ANBE) by enzyme histochemistry and with ferritin (Fer), α1-antitrypsin (AT), α1-antichymotrypsin (ACT), fibronectin (FN), HLA-DR, HLA-DP, Leu 10 and OKT 9 in immunohistochemical studies. MFH tumour cells did not immunostain with monocyte/macrophage markers (Leu M1, Leu M3, Mo 1, Mo 2 and Macrophage) although non-neoplastic histiocytes did react to these markers. In addition, granulation tissue, such as that found in sacral decubitus ulcers, was examined and the existence of a specific cell type called the “fibrohistiocytoid (FH) cell” was documented. The FH cell was short, spindle shaped and elliptical. Ultrastructurally, it had fragmented rER distributed in a branching pattern, dispersed free ribosomes, small mitochondria and a few dense bodies, but lacked diverse fused lysosomes and distinct pseudopodial cytoplasmic extensions. The FH cells reacted with AcP, alkaline phosphatase and ANBE but not with peroxidase using enzyme histochemistry and with Fer, AT, ACT, FN, HLA-DR, HLA-DP, Leu 10 and OKT 9 but not with monocyte/macrophage markers, C3d receptor, C3bi receptor in immunohistochemical studies. The FH cells had morphological, enzyme histochemical and immunohistochemical characteristics intermediate between fibroblasts and histiocytes. Similarities between MFH cells and the FH cells seen in chronic inflammation are discussed.
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  • 27
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    Virchows Archiv 415 (1989), S. 21-29 
    ISSN: 1432-2307
    Keywords: Alcoholic liver disease ; Ultrastructure ; Phagocytosis ; Cell shedding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Sinusoidal macrophages were studied by light and electron microscopy in 49 liver biopsies from alcohol-abusers with a variety of alcohol-related liver lesions or with near-normal livers. Changes were related to those in nearby hepatocytes. A reduction in the number of macrophages was noted in the more severely damaged livers. Hepatocytes formed blebs at their sinusoidal poles, and these protruded into the space of Disse and into the sinusoidal lumen. It is postulated that reduced phagocytic activity in the livers of patients with severe alcohol-related liver disease leads to increased shedding of hepatocellular material into the circulation. This may promote the development of autoimmune reactions directed against hepatocytes.
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  • 28
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    Archives of dermatological research 281 (1989), S. 35-39 
    ISSN: 1432-069X
    Keywords: Pseudo-Kaposi's sarcoma ; Bluefarb-Stewart syndrome ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary An ultrastructural study of the skin lesion of a young patient affected by pseudo-Kaposi's sarcoma of the Bluefarb-Stewart type (BSS) is reported. The neoplasm consisted of a proliferation of vascular structures mostly consisting of a solid bud of endothelial cells surrounded by a thinned and polystratified basement membrane and several pericytes. Both endothelial cells and pericytes were of normal ultrastructural appearance. Intervascular “stromal” cells were few and morphologically identified as macrophages and/or phagocytic fibroblasts. Masses of hemosiderin were detected outside the cells and in the macrophages, endothelial cells, and pericytes. Intracytoplasmatic crystalloid inclusions similar to those found in fetal endothelium and hemangiomas were observed in a few endothelial cells. These findings are different from those of previously reported cases of pseudo-Kaposi's sarcoma and may be helpful in distinguishing Kaposi's sarcoma from BSS. The role of immunodeficiency in the onset of BSS is discussed.
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  • 29
    ISSN: 1432-2013
    Keywords: Exercise ; Heart ; Mitochondria ; Oxygen uptake ; Respiration ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The relationship between maximal oxygen consumption rate ( $$\dot V{\text{O}}_{{\text{2max}}}$$ ) and mitochondrial content of skeletal muscles was examined in horses and steers (n=3 each). Samples of the heart left ventricle, diaphragm,m. vastus medialis, m. semitendinosus, m. cutaneous thoracicus andm. masseter, as well as samples of muscles collected in a whole-body sampling procedure, were analyzed by electron microscopy. $$\dot V{\text{O}}_{{\text{2max}}}$$ per kilogram body mass was 2.7× greater in horses than steers. This higher $$\dot V{\text{O}}_{{\text{2max}}}$$ was in proportion to the higher total volume of mitochondria in horse versus steer muscle when analyzed from the whole-body samples and from the locomotor muscle samples. In non-locomotor muscles, total mitochondrial volume was greater in horses than steers, but not in proportion to their differences in $$\dot V{\text{O}}_{{\text{2max}}}$$ . The $$\dot V{\text{O}}_{{\text{2max}}}$$ of the mitochondria was estimated to be close to 4.5 ml O2·ml−1 mitochondria in both species. It is concluded that in a comparison of a highly aerobic to a less aerobic mammalian species of similar body size, a higher oxidative potential may be found in all muscles of the more aerobic species. This greater oxidative potential is achieved by a greater total volume of skeletal muscle mitochondria.
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  • 30
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    Experimental brain research 76 (1989), S. 12-20 
    ISSN: 1432-1106
    Keywords: Distribution ; Ultrastructure ; Biopsy ; Catecholamines ; Interneurons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In freshly fixed biopsies of human cerebral cortex obtained at surgery, immunocytochemical staining with antibodies against tyrosine hydroxylase (the rate limiting biosynthetic enzyme for catecholamines) revealed, in addition to a dense axonal plexus, a population of immunoreactive cell bodies. The neuronal nature of these cells was ascertained by: i) the presence of a rich rough endoplasmic reticulum in the cell body and of synapses on the cell body and dendrites, and ii) the demonstration of the lack of reactivity with the astroglial marker, glial fibrillary acidic protein, in the tyrosine hydroxylase-immunoreactive cells. The tyrosine hydroxylase-immunoreactive neurons were found in all areas of cortex sampled, and were located almost exclusively in the infragranular layers. Most tyrosine hydroxylase-immunoreactive cells were bipolar and were vertically oriented, but a few had a multipolar or horizontal dendritic arbor. The dendrites of these cells were varicose and aspiny, and the axons were very thin. Tyrosine hydroxylase-immunoreactive neurons were reported to be present transiently in the developing mammalian cerebral cortex and only recently in cerebral cortex of mature mammalian brains. Internuncial neurons in the human cerebral cortex containing a catecholamine synthesizing enzyme would be significant, in particular considering that catecholamines are likely to be involved in some major mental disorders.
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  • 31
    ISSN: 1432-1106
    Keywords: Calcitonin gene-related peptide (CGRP) ; Bed nucleus of the stria terminalis ; Central nucleus of the amygdala ; Ultrastructure ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Light and electron microscopic analysis of calcitonin gene-related peptide (CGRP)-like immunoreactive (LI) terminals in the bed nucleus of the stria terminalis (BST) and the central nucleus of the amygdala (Ce) was carried out using the peroxidase-antiperoxidase method. CGRP-LI fibers were densely distributed in the dorsal subdivision of the lateral BST (BSTL) and the lateral and lateral capsular subdivisions of the Ce, where the CGRP-LI terminals formed symmetrical and asymmetrical axo-dendritic, and symmetrical axosomatic synapses. One of the most characteristic features of the CGRP-LI terminals was the presence of large, long boutons, each of which surrounded a cell soma and made many synaptic contacts. These findings suggest that CGRP exerts a significant influence on neurons in the BSTL and Ce.
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  • 32
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    European archives of oto-rhino-laryngology and head & neck 246 (1989), S. 169-172 
    ISSN: 1434-4726
    Keywords: Tympanic membrane ; Ultrastructure ; Sensory receptors ; Nerve endings
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Encapsulated nerve endings were found in both the subepidermal connective tissue and the lamina propria of a human tympanic membrane. The structure of the corpuscles was round or oval and contained a number of axon terminals with mitochondria and Schwann cell processes. Amorphous materials were present in the intercellular space. These features appear to be advantageous in transmitting mechanical forces on the capsule to the axon terminals and are comparable to the function of a mechanoreceptor. Resultant changes in the shape and stiffness of the tympanic membrane as the result of its dislocation indicate similar changes in the pressure on the corpuscle. The arrangement of the sensory corpuscles suggests that they may play a role in detecting pressure changes in the middle ear cavity.
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  • 33
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    European archives of oto-rhino-laryngology and head & neck 246 (1989), S. 56-60 
    ISSN: 1434-4726
    Keywords: Carbonic anhydrase ; Vestibular organ ; Guinea pig ; Histochemistry ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Carbonic anhydrase activity was demonstrated cytochemically on an ultrastructural level in the vestibular end organs of the guinea pig. Reaction product was found in the dark cells, transitional cells, cells of the planum semilunatum and supporting cells. In the dark cells, reaction product was observed in the cytoplasm as well as in the basal infoldings. Reaction product was also observed in the basal infoldings of the transitional cells and the cells of the planum semilunatum. The globular structures inside the supporting cells, transitional cells and the cells of the planum semilunatum were also surrounded by the reaction product. These findings suggest that carbonic anhydrase may have different functions, such as water and ion transport, respiration, nutrition and calcium carbonate deposition in the vestibular end organs.
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  • 34
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    Sexual plant reproduction 2 (1989), S. 193-198 
    ISSN: 1432-2145
    Keywords: Polymorphism ; Ultrastructure ; Pollen grains ; Canna indica L ; Tannin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.
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  • 35
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    Sexual plant reproduction 2 (1989), S. 154-166 
    ISSN: 1432-2145
    Keywords: Helianthus annuus ; Unfertilized ovule culture ; Parthenogenesis ; Ultrastructure ; Proembryo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Electron microscope studies have been conducted on the parthenogenesis induced by in vitro culture of unfertilized ovules of sunflower (Helianthus annuus). In comparison with the state of the egg prior to inoculation, some eggs 5 days after culture show striking ultrastructural changes, which include, among others, nuclear migration, an increase in the number and activity of the organelles, a loss of polarity and wall formation at the chalazal end of the cell. Most of these changes are similar to those that occur normally in the zygote, indicating that parthenogenic development has been triggered in these eggs. Such eggs have been termed activated and are presumed to be capable of undergoing parthenogenesis. The parthenogenic proembryos which result share some features in common with zygotic proembryos. In addition, some parthenogenic proembryos exhibit unique properties not found in zygotic proembryos. These include embryos that consist of two parts differing markedly in density, an inversion of polarity, the frequent occurrence of autophagic vacuoles, the thickening of cell walls, a centripetal growth mode of wall formation, the appearance of an incomplete cell wall, free nuclear division, amitosis and degeneration. We believe that these ultrastructural peculiarities are the effects of in vitro culture.
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  • 36
    ISSN: 1435-5604
    Keywords: Intercellular communication ; Gap junction ; Calcification ; Collagen gel ; Ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary To analyze the mechanism of initiation of cell-mediated calcification in hard tussue and its relationship to the frequency of gap junctions, enzymatically isolated cells from fetal rat calvaria cultured in collagen gels were observed ultrastructurally over a time course. Calcification was observed at 2–3 weeks after the initiation of culture when the seeding cellularity and the concentration of β-glycerophosphate were sufficiently high. In the collagen gels, round cells (R), spindle or stellate cells (S), and fat cells (F) were characterised morphologically. The ultrastructural features of initial calcification could be classified into 4 subtypes: 1) a large mass greater than 10 µm in diameter (Type I), 2) deposition associated with dead R cells or matrix vesicles (Type II), 3) intracellular deposition (Type III), and 4) other than Types I–III (Type IV). Type II was the most frequent (44.5%) and Type III was the least (6.8%). Gap junction was observed frequently between 1) R cells, 2) S cells, 3) between R cells and S cells. The frequency of gap junctions in collagen gels decreased statistically (X2-test; p〈0.001), when calcification was initiated. This cell culture system can be regarded as a useful model to analyze the initiation of cell mediated calcification in hard tissue. Gap junctions might function in cell communication and a decrease in their numbers could lead to cell death and, subsequently to calcification.
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  • 37
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    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 38
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 1-7 
    ISSN: 0887-3585
    Keywords: helix stabilization ; helix dipole ; charged group ; pH titration ; electrostatic interaction ; hydrogen bonding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interactions between the α-helix peptide dipoles and charged groups close to the ends of the helix were found to be an important determinant of α-helix stability in a previous study.1 The charge on the N-terminal residue of the C-peptide from ribonuclease A was varied chiefly by changing the α-NH2 blocking group, and the correlation of helix stability with N-terminal charge was demonstrated. An alternative explanation for some of those results is that the succinyl and acetyl blocking groups stabilize the helix by hydrogen bonding to an unsatisfied main-chain NH group. The helix dipole model is tested here with peptides that contain either a free α-NH3+ α-COO- groups, and no other charged groups that would titrate with similar pKa's. This model predicts that α-NH3α-COO- groups are helix-destabilizingand that the destabilizing interactions are electrostatic in origin. The hydrogen bonding model predicts that α-NH3 and α-COO- groups are not themselves helix-destabilizing, but that an acetyl or amide blocking group at the N- or C- terminus, respectively, stabilizes the helix by hydrogen bonding to an unsatisfied main-chain NH or CO group.The results are as follows: (1) Removal of the charge from α-NH3 and α-COO- groups by pH titration stabilizes an α-helix. (2) The increase in helix stability on pH titration of these groups is close to the increase produced by adding an acetyl or amide blocking group. (3) The helix-stabilizing effect of removing the charge from α-NH3 and α-COO- groups by pH titration is screened by increasing the NaCl concentration, and therefore the effect is electrostatic in origin. (4) Replacing the C-terminal amide blocking group with a methylester blocking group, which cannot donate a hydrogen bond, causes little change in helix stability.
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  • 39
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 8-12 
    ISSN: 0887-3585
    Keywords: ribonuclease A ; protein deamidation ; protein conformation ; disulfide bonds ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determine. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds.Deamidation of the labile Asn-67 residue of RNase A was followedelectrophoretically and chromatographically. At 80°C, similar rates of deamidation were observed for the disulfidebonded form, which is thermally unfolded, and the reduced form. At 37°C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the β-turn of residues 66-68.
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  • 40
    ISSN: 0887-3585
    Keywords: template-assembled synthetic protein (TASP) ; 4-helix bundle ; β-barrel structure ; protein de novo design ; peptide synthesis ; peptide conformation ; orthogonal protection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a β-barrel as two folding “domains” is described. For the de novo design of proteins, amphiphilic helices (α) and β-sheets (β) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intarmolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4α)(4β). The design of this new macromolecule was assisted by computer modeling, which suggested a low-energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid-phase synthesis of the “two-domain” TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.
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  • 41
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 22-37 
    ISSN: 0887-3585
    Keywords: sequence homology ; tertiary structure prediction ; molecular dynamics ; energy minimization ; hydrophobic interactions ; aromatic ring-ring interactions ; salt bridges ; calcium binding ; thermoactinomyces vulgaris ; extracellular protease ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, Subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three-dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserve structural core and less well in seven loop regions involving insertions and deletions relative to Subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular case of thermostability we confront hypotheses erived by calculation of the details of interatomic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main cause of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.
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  • 42
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 47-65 
    ISSN: 0887-3585
    Keywords: crystal growth ; helical conformation ; repetitive octapeptide ; icelike molecular surface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The active conformation of an icenucleation protein, whose major portion consists of a long polypeptide segment of nearly repetitive octapeptides, is predicted by the analyses of conformational energy and the mechanism of crystal growth. The protein ideally has an exact octapeptide repetition and is assumed to have a helical conformation. The present study searched for low-energy helical conformations and each of the obtained low-energy conformations examined as to whether it has a surface structure that can promote crystal formation. Two conformations obtained were good candidates for an ice nucleus. Both were found to have on their surfaces an arrangement of hydrogen-bonding sites, which fits well with those of hydrogen bonds in hexagonal ice crystal. Further, one of the two conformations had a hexagonal conformational symmetry consistent with the hexagonal ice crystal structure. The other conformation had apentagonal conformational symmetry that could enable the growth of an ice crystal-dendritic polycrystalline snow crystal-which grows on metastable cubic ice.
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  • 43
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 38-46 
    ISSN: 0887-3585
    Keywords: reconstitution ; membrane protein folding/unfolding ; pH effects ; differential scanning calorimetry ; circular dichroism ; electron microscopy ; HPLC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Thermal unfolding experiments on bacteriorhodopsin in mixed Phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 ± 13 Å in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation. We conclude that the tertiary structure of the bacteriorhodopsin monomer is essentially the same in micelles and the purple membrane. On the other hand, in the synthetic mixed micelle system, the packing between the nonnative amphiphiles and bacteriorhodopsin is probably not optimal, protein-protein interactions have been lost, and thehelical packing may be looser because the crystalline lattice is absent. Itis likely that a combination of these effects leads to the decreased stability of micellar bacteriorhodopsin.
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  • 44
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 78-92 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 1 Ill.
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  • 45
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    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 46
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 93-95 
    ISSN: 0887-3585
    Keywords: folding intermediate ; molten globule state ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two-step folding mechanism. These results are summarized and interpreted according to the “molten globule” model. The molten globule state of bGH is characterized as a folding intermediate which largely a-helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent-exposed hydrophobic surface along helix 106127 that readily leads association.
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  • 47
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 66-77 
    ISSN: 0887-3585
    Keywords: subunit interactions ; allosteric regulation ; solvent accessibility ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Although ionizable groups are known to play important roles in the assembly, catalytic, and regulatory mechanisms of Escherichia coli aspartate transcarbamylase, these groups have not been characterized in detail. We report the application of static accessibility modified Tanford-Kirkwood theory to model electrostatic effects associated with the assembly of pair of chains, subunits, and the holoenzyme. All of the interchain interfaces except R1-R6 are stabilized by electrostatic interactions by -2 to -4 kcal-m-1 at pH 8. The pH dependence of the electrostatic component of the free energy of stabilization of intrasubunit contacts (C1-C2 and R1-R6) is qualitatively different from that of intersubunit contacts (C1-C4, C1-R1, and C1-R4). This difference may allow the transmission of information across subunit interfaces to be selectively regulated. Groups whose calculated pK or charge changes as a result of protein-protein interactions have been identified and the results correlated with available information about their function. Both the 240s loop of the c chain and the region near the Zn(II) ion of the r chain contain clusters of ionizable groups whose calculated pK values change by relatively large amounts upon assembly. These pK changes in turn extend to regions of the protein remote from the interface. The possibility that networks of ionizable groups are involved in transmitting information between binding sites is suggested.
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  • 48
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    Proteins: Structure, Function, and Genetics 5 (1989), S. i 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 49
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 97-103 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; spin glass ; conformational substates ; conformational heterogeneity ; hierarchy in dynamics ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A computer experiment of protein dynamics is carried out, which consists of two steps: (1) A Monte Carlo simulation of thermal fluctuations in the native state of a globular protein, bovinepancreatic trypsin inhibitor; and (2) a simulation of the quick freezingof fluctuating conformations into energy minima by minimization of the energy of a number of conformations sampled in the Monte Carlo simulations sampled in the Monte Carlo simulation. From the analysis of results of the computer experiment is obtained the following picture of protein dynamics:multiple energy minima exist in the native state, and they are distributedin clusters in the conformational space. The dynamics has a hierarchical structure which has at least two levels. In the first level, dynamics is restricted within one of the clusters of minima. In the second, transitions occur among the clusters. Local parts of a protein molecule, side chains and local main chain segments, can take multiple locally stable conformations in the native state. Many minima result from combinations of these multiple local conformations. The hierarchical structure in the dynamics comes from interactions among the local parts. Protein moleculeshave two types of flexibility, each associated with elastic and plastic deformations, respectively.
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  • 50
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 104-112 
    ISSN: 0887-3585
    Keywords: conformational fluctuations ; conformational heterogeneity ; conformational energy ; hierarchical structure ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Conformational fluctuations in a globular protein, bovine pancreatic trypsin inhibitor, in the time range between picoseconds and nanoseconds are studied by a Monte Carlo simulation method. Multipleenergy minima are derived from sampled conformations by minimizing their energy. They are distributed in clusters in the conformational space. A hierarchical structure is observed in the simulated dynamics. In the time range between 10-14 and 10-10 seconds dynamics is well represented by a superposition of vibrational motions within an energy well with transitions among minima within each cluster. Transitions among clusters take place in the time range of nanoseconds or longer.
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  • 51
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 113-124 
    ISSN: 0887-3585
    Keywords: conformational fluctuations ; Monte Carlo simulation ; conformational energy ; conformational heterogeneity ; side chain conformation ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An analysis is carried out of differences in the minimum energy conformations obtained in the previous paper by energy minimization starting from conformations sampled by a Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatictrypsin inhibitor. Main conformational differences in each pair of energy minima are found usually localized in several side chains and in a few localmain chain segments. Such side chains and local main chain segments are found to take a few distinct local conformations in the minimum energy conformations. Energy minimum conformations can thus be described in terms of combinations of these multiple local conformations.
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  • 52
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 125-131 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; plastic deformation ; conformational heterogeneity ; hierarchy in dynamics ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Differences in atom packing are studied in the minimum energy conformations derived from the record of the Monte Carlo simulation of conformational fluctuation in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations observed among the minima which are found in the previous paper are accompanied by rearrangement of atom packing. Spatial locations of the local deformations in the three-dimensional folded structure are also studied. It is foundthat the local deformations are distributed in space in several clusters inthe folded structure. The size and location of the clusters characterize the respective fluctuations of the first and the second levels observed in the simulation. In the fluctuations of the first level local deformations, each of which usually involves a few side chains and one main chain local segment, are thermally exited independently of each other near thesurface of the molecule. The observed fluctuation of the second level involves a cooperative deformation involving many side chains and local main chain segments all in one cluster, which goes though the core of the molecule. The collective local deformations observed both in the first and second levels are plastic in the sense that they are accompanied with rearrangement of atom packing.
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  • 53
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 139-148 
    ISSN: 0887-3585
    Keywords: protein architecture ; packing ; evolutionary relationships ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: α/β barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight-stranded β-sheets and the packing of the residues at the center of the barrel. The Packingin thisregion is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at someof the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues fromthe second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even-numbered strands that form the central layer, and side chains from the oddnumbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed β barrel.
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  • 54
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 132-138 
    ISSN: 0887-3585
    Keywords: protein conformation ; dynamics ; Monte Carlo simulation ; conformational energy ; minimization ; hierarchy in dynamics ; conformational heterogeneity ; flexibility ; trypsin inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Distances between centers of gravity of individual residues are compared among the minimum energy conformations derived from the recordof the Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatic trypsin inhibitor. It is found that local deformations originating from the multiplicity of localconformations cause deformations of the whole structure of the molecule in various ways, which can be classified into two types. Type 1:When a local deformation occurs in a region consisting of a few residues near the surfaceof the molecule, the whole shape of the molecule responds by deforming elastically. The magnitude of this deformation is in the range of thermalfluctuations calculated by the harmonic approximation around a singleminimum. Type 2: We have observed one case belonging to the second type in which local deformations occur cooperatively in an extended region. This regiongoes across the whole molecule and divide the remaining parts into two. Atom packing changes in and around the extended region of local deformations. For this reason deformation in this region is plastic. Relative locationand orientation between the divided two parts change very much. Deformationof the whole shape in this case, associated with the plastic deformationin an extended region, demonstrates that protein molecules have a flexibility beyond the harmonic limit.
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  • 55
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 149-155 
    ISSN: 0887-3585
    Keywords: solution scattering ; low-angle scattering ; spherical averaging ; spherical harmonics ; spherical Fourier transform ; bound water ; solvent structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: If one expands the structure factor equation in spherical coordinates, rotational averaging of the molecular Fourier transform, which leads directly to the solution scattering profile, is greatly simplified. It becomes a projection in the polar and azimuthal angular variables. The profile is given by The index j runs over all atoms; r, θ, φ are atomic coordinates and ε and N are constants; the Ym,n are complex spherical harmonics, and Jn are spherical Bessel functions; R = 2 sin θ/λ. The effects of solvent have been modeled by subtracting from each protein atom a properly weighted water. Hydrogens have been included by using scattering curves fj derived from the spherical averaging ofprotein atoms with their attached hydrogens. This approach may also be satisfactory for neutron scattering. Published scattering profiles2 for lysozyme and BPTI have been accurately matched in less than one-tenth the time required by other methods. Separate, adjustable temperature factors for the protein, solvent waters, and bound watersare used, and appear to be needed. In the case of BPTI, as suggested by NMR observations, the observed diffraction pattern was much better accounted for by including only 4 tightly bound waters rather than the roughly 60 seen by crystallography.
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  • 56
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 156-165 
    ISSN: 0887-3585
    Keywords: retrovirus integrase ; circular dichroism ; homologous proteins ; secondary structural predictions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The retrovirus integrase (IN) protein is essentialfor integration of viral DNA into host DNA. The secondary structure of thepurified IN protein from avian myeloblastosis virus was investigated by bothcircular dichroism (CD) spectroscopy and five empirical prediction methods. The secondary structures determined from the resolving of CD spectra through a least-squares curve fitting procedure were compared with those predicted from four statistical methods, e.g., the Chou-Fasman, arnier-Osguthorpe-Robson, Nishikawa-Ooi, and a JOINT scheme which combined all three of these methods, plus a pure a priori one, the Ptitsyn-Finkelstein method. Among all of the methods used, the Nishikawa-Ooiprediction gave the closest match in the composition of secondary structureto the CD result, although the other methods each correctly predictedoneor more secondary structural group. Most of the α-helix and β-sheet states predicted by the Ptitsyn-Finkelstein methodwere in accord with the Nishikawa-Ooi method. Secondary structural predictions by the Nishikawa-Ooi method were extended further toinclude IN proteins from four phylogenetic distinct retroviruses. The structuralrelationships between the four most conserved amino acid blocks of these IN proteins were compared using sequence homology and secondary structure predictions.
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  • 57
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 166-169 
    ISSN: 0887-3585
    Keywords: α-crystallin ; enthalpy ; entropy of solution ; light scattering ; second virial coefficient ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Light scattering studies were performed on bovine α-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of α-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of α-crystallin are positive. The Flory thetatemperature was found to be 271 K.
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  • 58
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 170-182 
    ISSN: 0887-3585
    Keywords: modeling ; flavodoxin ; structure prediction ; side chains ; database ; structure analysis ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The tertiary structure of flavodoxin has been model build from only the X-ray crystallographic α-carbon coordinates. Main-Chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 Å across all atoms to the native structure. Regular secondary structural elements were modeledmore accurately than other regions. About 40%of the ξ1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions.The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contact, and hydrogen-bonding patterns could identifypoorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.
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  • 59
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    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 60
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 202-210 
    ISSN: 0887-3585
    Keywords: enhanced stability ; λCro ; genetic suppression ; intracellular proteolysis ; antibody screen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A mutant Cro protein, which bears the Ile-30→ Leu substitution, is thermally unstable and degraded more rapidly than wildtype Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12°C to 14°C. These amino acid substitutions affect residues 16 and 26, which are substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild-type Cro.
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  • 61
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 183-201 
    ISSN: 0887-3585
    Keywords: crystallographic refinement ; restrained least-squares refinement ; Konnert-Hendrickson refinement ; phosphodiesterase ; protein structure ; enzyme mechanism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3′,5′-biophosphate (pdTp) has been refined by stereochemically restrained leastsquares minimization to a crystallographic R value of 0.161 at Å resolution. The estimated root-mean-square (rms) error in the coordinates in 0.16 Å. The final model comprises 1082 protein atoms, onecalcium ion, the pdTp molecule, and 82 protein atoms, onecalcium ion, the pdTp molecule, and 82 solvent water molecules;it displays an rms deviation from ideality of 0.017 Å for bond distances and 1.8° for bond angles.The mean distance between corresponding α carbons in the refined and unrefined structures is 0.6 Å we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket.The details of the calcium liganding and solvent structure in the activesite are clearly shown in the final electron density map. The structure ofthe catalytic site is consistent with mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry-related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that enzyme and substrate.
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  • 62
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 211-217 
    ISSN: 0887-3585
    Keywords: tyrosin ; mutant protein ; amino acid substitution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: CD spectra in the aromatic region of a series of the mutant α-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25°C. The measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of thewild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CDvalues of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for oneband at 276.5 nm. these results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residuesat position 49.
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  • 63
    ISSN: 0887-3585
    Keywords: yeast hexokinase II ; dimerization ; in vivo functions ; glucose repression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form).This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short from protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similarto wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form ofthe enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminalamino acids of hexokinase II are not required in vivo either for phosporylation of hexoses or for glucose repression.
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  • 64
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 233-247 
    ISSN: 0887-3585
    Keywords: protein folding ; crystallographic data base ; structural analysis ; computer program system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: PKB is a computer program system that combines a data base of three-dimensional protein structures with a series of algorithms for pattern recognition, data analysis, and graphics. By typing relatively simple commands the user may search the data base for instances of a structural motif and analyze in detail the set of individual structures that are found. The application of PKB to the study of protein folding is illustrated in three examples. The first analysis compares the conformations observed for a short sequential motif, sequences similar to the cell-attachment signal Arg-Gly-Asp. The second compares sequences observed for a conformational motif, a 16-residue βαβ unit. The third analysis considers a population of substructures containing ion-pair interaction, examining the relationship offrequency of occurrence to calculated electrostatic energy.
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  • 65
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 224-232 
    ISSN: 0887-3585
    Keywords: effective pore's radius ; α-ketoglutarate dehydrogenase complex ; branched chain α-keto acid dehydrogenase complex ; electron microscopy ; multienzyme complex ; two-dimensional ; electrophoresis ; multienzyme complex ; aggregation of Pyruvate dehydrogenase complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two-dimensional agarose gel electrophoresis (2d-AGE) (Serwer, P. et al. Anal. Biochem. 152: 339-345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10-30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α-ketoglutarate (α-KGDC), and the branched chain α-keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross-linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d-AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d-AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.
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  • 66
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    Proteins: Structure, Function, and Genetics 5 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 67
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 248-257 
    ISSN: 0887-3585
    Keywords: subunit interactions ; icosahedral capsid ; electrostatic potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The role of the electrostatic interactions in the stability of the icosahedral β60 capsid of heavy riboflavin synthase from Bacillus Subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interaction agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between β-subunits forming dimers, which connect the relatively stable pentamers in the β-60 capsid. The release of the ligand causesareduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due tothe titratable groups and α-helix macrodipoles has been calculated on the basic of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polardistribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of the positive potential “channels” that coincide with the channels constituted by the pentameric and trimeric β-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.
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  • 68
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 259-265 
    ISSN: 0887-3585
    Keywords: cDNA expression ; deletion mutagenesis ; zymogen activation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cellsto secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 266-270 
    ISSN: 0887-3585
    Keywords: crystallization ; purification ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 × 0.3 × 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which in enzymatically active upon dissolution. These crystals belong to the monoclinic space group P21 and have unit cell parameters a =114.5 Å, b=139.6 Å, c=125.7 Å, β=98.1° Self-rotation function studies indicate that there are three molecules per asymmetricunit. The crystals diffract to at least 3.0 Å resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.
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  • 70
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 281-288 
    ISSN: 0887-3585
    Keywords: Hu protein ; integration host factor ; transcription factor 1 ; DNA bending protein ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high-resolution structure of HU, we propose a model for thisinteraction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.
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  • 71
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 271-280 
    ISSN: 0887-3585
    Keywords: antifluorescyl monoclonal antibody ; high-affinity binding site ; effects of MPD on hapten binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 Å resolution by molecularreplacement methods. The starting model was the refined 2.7 Å structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecules relative to other members of an idiotypic family of monoclonalantifluorescyl antibodies. The phenyl carboxyl group of fluorescein appearsto be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domainsjust below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.
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  • 72
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 289-312 
    ISSN: 0887-3585
    Keywords: aconitase ; iron-sulfur enzyme ; crystal structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The crystal structure of the 80,000 Da Fe—S enzyme aconitase has been solved and refined at 2.1 Å resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Associationof the larger C-terminal domain with the first three domains createsan extensive cleft leading to the Fe—S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe—S cluster and a bound SO42-ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well asseveral bound water molecules. Three of these side chains reside on a threeturn 310 helix in the first domain. The SO42-ion is bound 9.3 Å from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln rsidues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.
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  • 73
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 313-321 
    ISSN: 0887-3585
    Keywords: protein ; electron transfer ; molecular dynamic simulations ; dielectric ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Electrostatic calculations have been carried out on a number of structural conformers of tuna cytochrome c Conformers were generated using molecular dynamics simulations with a range of solvent simulating, macroscopic dielectric formalisms, and one solvent model that explicitly included solvent water molecules. Structures generated using the lowest dielectric models were relatively tight, with-side chains collapsed on the surface, while those from the higher dielectric modelshad more internal and external fluidity, with surface side chains exploring a fuller range of conformational space. The average structure generated with the explicitly solvated model corresponded most closely with the crystal structure. Individual pK values, overall titration curves, and electrostatic potential surfaces were calculated for average structures and along each simulation. Differences between structural conformers within each simulation give rise to substantial changes in calculated local electrostatic interactions, resulting in pK value fluctuations for individual sites in the protein that very by 0.3-2.0 pK units from the calculated time average. These variations are due to the thermal side chain reorientations that produce fluctuations in charge site separations. Properties like overall titration curves and pH dependent stability are not as sensitive to side chain fluctuations within a simulation, but there are substantial effects between simulation due to markeddifferences in average side chain behavior. These findings underscore the importance of proper dielectric formalism in molecular dynamics simulations when used to generate alternate solution structures from a crystal structure, and suggest that conformers significantly removed from the averagestructure have altered electrostatic properties that may prove important inepisodic protein properties such as catalysis.
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  • 74
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 322-336 
    ISSN: 0887-3585
    Keywords: sequence conservation ; exon ; gene duplication ; protein folding ; structure-function ; X-ray structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key β-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of uperoxide dismutation. The β-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the β-strands and at both termini. Thus, the β-barrel with only four invariant residues is apparently over determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility ofthe Greek key β-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.
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  • 75
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    Proteins: Structure, Function, and Genetics 6 (1989) 
    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 6 (1989), S. 1-19 
    ISSN: 0887-3585
    Keywords: virus crystallography ; molecular dynamics ; hydrophobic pockets ; antiviral agents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: X-Ray diffraction data have been obtained for nine related antiviral agents (“WIN compounds”) while bound to human rhinovirus 14 (HRV14).These compounds can inhibit both viral attachment to host cells and uncoating. To calculate interpretable electron density maps it was necessary to account for (1) the low (∼60percnt;) occupancies of these compounds in the crystal, (2) the large (up to 7.9 Å) conformational changes induced at the attachment site, and (3) the incomplete diffraction data. Application of a density difference map technique, which exploits the 20-fold noncrystallographic redundancy in HRV14, resulted in clear images of the HRV14:WIN complexes. A real-space refinement procedure was used to fit atomic models to these maps.The binding site of WIN compounds in HRV14 is a hydrophobic pocket composed mainly from residues that form the β-barrel of VP1. Among rhinoviruses, the residues associated with the binding pocket are far more conserved than external residues and are mostly contained within regular secondary structural elements. Molecular dynamics simulations of three HRV14:WIN complexes suggest that portions of the WIN compounds and viral protein near the entrance of the binding pocket are more flexible than portions deeper within the β-barrel.
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  • 77
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 337-354 
    ISSN: 0887-3585
    Keywords: computer simulation ; fluctuations in proteins ; secondary structural dynamics ; lysozyme ; protein-substrate complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, β-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and β-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of theloop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions withthe substrate. Moreover, part of a loop and a 310 helix (residues of 67-88) not in contact with the substrate showeda marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuationsof the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain φ and ψ angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place eitherabruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 Å rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface areaand the dynamics of the different structural elements.
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  • 78
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    Proteins: Structure, Function, and Genetics 5 (1989), S. 355-373 
    ISSN: 0887-3585
    Keywords: protein ; structure ; prediction ; primary ; secondary ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of Cα coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set ofstandard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can “replace” about 76% ofall hexamers in well-refined known proteins with an error of less than 1 Å, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.
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  • 79
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 20-31 
    ISSN: 0887-3585
    Keywords: ribonuclease ; active site ; conformational change ; protein-nucleic ; acid interactions ; fluorescence depolarization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulations were performed on free RNase T1 and the 2′GMP-RNase T1 complex in vacuum and with water in the active site along with crystallographically identified waters, allowing analysis of both active site and overall structural and dynamics changes due to the presence of 2′GMP. Difference in the active site include a closing in the presence of 2′GMP, which is accompanied by a decrease in mobility of active site residues. The functional relevance of the active site fluctuations is discussed. 2′GMP alters the motion of Tyr-45, suggesting a role for that residue in providing a hydrophobic environment for the protein-nucleic acid interactions responsible for the specificity of RNase T1. The presence of 2′GMP causes a structural change of the C-terminus of the α-helix, indicating the transmission of structural changes from the active site through the protein matrix. Overall fluctuations of both the free and 2′GMP enzyme forms are in good agreement with X-ray temperature factors. The motion of Trp-59 is influenced by 2′GMP, indicating difference in enzyme dynamics away from the active site, with the calculated changes following those previously seen in time-resolved fluorescence experiments.
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  • 80
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 46-60 
    ISSN: 0887-3585
    Keywords: macromolecular conformation ; protein folding ; helical axis ; secondary structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinated of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry. This algorithm has been implemented in a computer program named P-Curve. Several examples of its possible applications are discussed.
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  • 81
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 61-69 
    ISSN: 0887-3585
    Keywords: peptides ; hormones ; amphipathic helix ; hydrophobic moment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Eisenberg's helical hydrophobic moment (〈μH〉) algorithm was applied to the analysis of the primary structure of amphipathic α-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue 〈μH〉 for the amphipathic helical and control peptides were 0.46 (±/-0.07) and 0.33 (0.07) (P+0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular 〈μH〉 was plotted vs 〈μH〉. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic α helix at residues 4-21. This computer-based method may enable rapid identification of peptide of the amphipathic α-helix class.
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  • 82
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 32-45 
    ISSN: 0887-3585
    Keywords: long range truncation ; molecular dynamics ; myoglobin ; truncation effects ; protein electrostatics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: This paper considers the effects of truncating long-range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long-range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom-atom truncation based on distance; (4) simple atom-atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group-group truncation based on distance; and (6) simple group-group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates of a no cutoff simulation and from the X-ray structure of the protein. The simulations in which long-range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 Å, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5-9 Å range gives reasonable results for a distance-dependent dielectric, but not using a constant dielectric. Both the atom-atom and group-group truncation methods based on distance shows large rms deviations and fluctuation for short cutoff distance, while for cutoff distance of 11 Å or greater, reasonable results are achieved. Although comparison of these to distance-based truncation methods show surprisingly larger rms deviations for the group-group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom-atom or group-group list-based simulations generally appear to be less stable than the distance-based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.
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  • 83
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    Proteins: Structure, Function, and Genetics 6 (1989) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 84
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 70-85 
    ISSN: 0887-3585
    Keywords: Protein electrostatics ; protein kinases ; effector protein ; calciumbinding protein ; α-helix ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain α-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-ldependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charges in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.
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  • 85
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 87-103 
    ISSN: 0887-3585
    Keywords: molten globule state ; protein folding ; protein denaturation ; structural intermediate ; activated state of folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 6 Ill.
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  • 86
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 104-127 
    ISSN: 0887-3585
    Keywords: lac repressor; lac operator ; lac headpiece-operator complex ; protein-DNA specificity ; molecular dynamics ; computer simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complexsatisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator based on many direct- and water-mediated hydrogenbonds and nonpolar contacts which allow the formation of a tight complex. Nostable hydrogen bonds between side chains and bases and found, while specific contacts occur between both nonpolar groups and, to a lesserextent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, andchemically induced dynamic nuclear polarization (CIDNP) data.
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  • 87
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 128-138 
    ISSN: 0887-3585
    Keywords: DNA repair ; ultravioletlight ; pyrimidine dimers ; glycosylase ; apurinic/apyrimidinic endonuclease ; DNA repair enzymology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Previous structure/function analyses of the DNA repairenzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has beeninvestigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changesof Trp and Phe and more dramatic changes of Ser- a stop, codon, deletion of the codon or introduction of a frameshift. Both changesto the aromatic amino acids resulted in proteins which accumulated will in E. coli and not only significantly enhanced the UV survival of repair, deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129 → Trp and Tyr-129 → Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129 → Phe and Tyr-129 → Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129 → Phe mutant and to 20% that of wild type for the Tyr1-29 → Trp mutant.
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  • 88
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    Proteins: Structure, Function, and Genetics 6 (1989), S. 155-167 
    ISSN: 0887-3585
    Keywords: protein structure comparison ; dihedral angles ; protein conformation ; hemoglobin structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes Δt, the root mean square difference in φ and ψ torsion angles over a small number of amino acids (typically 3-5). Using this algorithm, large number of protein substrates comparisons were feasible. The parameter Δt was sensitive to variations in local protein conformation, and it correlates with Δr, the root mean square deviation in atomic coordinates. Values for Δt were obtained that define similarity thresholds, which determine whether two substructure are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of Δt due to measurement noise fromcomparisons of independently refined coordinates of the same protein. A sample distribution of Δt from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons large numbers of nonmatching comparisons. Unlike methods based on Cα atoms alone, Δt was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologus proteins by Δt showed that the active site torsion angles are highly conserved. The Δt method was applied to the α-chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.
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  • 89
    ISSN: 0887-3585
    Keywords: retrovirus ; bacterial expression ; high-performance liquid chromatography ; NH2- and COOH-terminal sequence analysis ; kcat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala-13 to Gly-185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short-lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species isonly partially processed, and it, a 13 kDA intermediate, and the mature 11 kDA enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2- and COOH-terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2≅ 9 min) processes itself into a species with a mass of ∼13kDa and a species with a mass of ∼11 kDa. Both of these latter species can be separated by RP-HPLC, have the NH2-terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP-HPLC and SDS poly acrylamide gel electrophoresis. On extended incubation at 4°C at either neutral or acidic pH all species of the proteinexhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989) 
    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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  • 91
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 215-215 
    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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  • 92
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 168-192 
    ISSN: 0887-3585
    Keywords: salt bridge ; ab initio ; calcium ; magnesium ; carboxylate ; phosphate ; ammonium ; guanidinium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Equilibrium geometries and binding energies of model “salt” or “ion” bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split-valence basis set. For the model compounds studied we find that the ab initio geometries are in remarkably goodagreement with the molecular mechanics geometries.Several Calculations werealso performed using diffuse fractions. The formate anion binds these modelcations more strongly than does dimethyl phosphate, while the organiccation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies togive useful insight into the details energies to give useful insight into the details of salt bridges in biological systems.
    Additional Material: 16 Ill.
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  • 93
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 217-221 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: No abstract.
    Type of Medium: Electronic Resource
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  • 94
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 222-230 
    ISSN: 0887-3585
    Keywords: G proteins ; p21ras ; GTPase ; cholera toxin ; GTPase-activating protein ; amino acid sequence ; protein structure ; conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The functions of G proteins - like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras) - depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein α-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the α-chain of Gs (αs) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of αs in human pituitary tumors inhibit-the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in αs that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of the GTPase inhibiting mutations in αs occurs in the codon for an ARG residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by αs. This Arg residue is located in a domain of αs not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein. Owing to their inherited similarities of structure and function, what we learn about αs, p21ras, or EF-tu as individual molecules helps us to understand crucial functions of other members of the super-family.
    Additional Material: 2 Ill.
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  • 95
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 193-209 
    ISSN: 0887-3585
    Keywords: protein folding ; simulated annealing ; empirical potentials ; Monte Carlo dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The current work describes a simplified representation of protein structure with uses in the simulation of protein folding. The model assumes that a protein can be represented by a freely rotating rigid chain with a single atom approximately the effect of each side chains. Potentials describing the attraction or repulsion between different types of amino acids are determined directly from the distribution of amino acids in the database of known protein structures. The optimization technique of simulated annealinghas been used to dynamically sample the conformations available to this sample model, allowing the protein to evolve from an extended, random coil into a compact globular structure. Many characteristics expected of true proteins, such as the sequence-dependent formation of secondary structure, the partitioning of hydrophobic residues, and specific disulfide, suggestion the model may accurately simulate the folding process.
    Additional Material: 8 Ill.
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  • 96
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 231-239 
    ISSN: 0887-3585
    Keywords: phosphotyrosine linkage ; protein-DNA transesterification ; enzyme mechanism ; DNA-protein covalent complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Tyrosine 319 of E. coli topoisomerase I is shown to be the activesite tyrosine that becomes covalently attached to a DNA 5′ phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme. The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA-linked tryptic peptide. Site-directed mutagenesis converting Tyr-319 to a serine or phenylalanine completely inactivates the enzyme. The structure of the enzyme andits catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available information.
    Additional Material: 4 Ill.
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  • 97
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 240-248 
    ISSN: 0887-3585
    Keywords: substrate-assisted catalysis ; serine protease ; fusion proteins ; site-directed mutagenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN′, which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wildtype subtilisin BPN′ is greatly restricted by substitution of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J. A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.
    Additional Material: 4 Ill.
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  • 98
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 249-258 
    ISSN: 0887-3585
    Keywords: DNA binding domain ; etheno-M13 DNA ; single-stranded DNA affinity chromatography ; proteolytic fragments ; truncated topoisomerase ; protein-DNA interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme.
    Additional Material: 5 Ill.
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  • 99
    ISSN: 0887-3585
    Keywords: β-adrenergic recepor ; chimeric proteins ; receptor subtypes ; ligand binding ; protein structure-function ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pharmacological analysis of ligand binding to the β-adrenergic receptor (βAR) has revealed the existence of two distinct receptor subtypes (β1 and β2) which are the products of different genes. The predicted amino acid sequence of the β1 and β2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human β1 and hamster β2 receptors. Analyses of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the βAR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacement of regions of the hamster β2AR with the analogous regions from the avian β1AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the β1 and β2 receptors.
    Additional Material: 4 Ill.
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  • 100
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 6 (1989), S. 275-283 
    ISSN: 0887-3585
    Keywords: mutagenesis ; structure-function relationships ; enzymatic catalysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 β-lactamase to assess the role of this site in modulating differences in specificity of β-lactamases for penams vs. cephams as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.1,2) Screening of all 19 possibles mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinnetic analysis showns that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RETM-1 β-lactamase and lower by about 5°C the tempreature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most intresting aspect of these results is that two quite disparate amino acids, theronine and asparagine, when intorduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.
    Additional Material: 3 Ill.
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