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  • 1990-1994  (2.648)
  • 1990  (2.648)
  • Life and Medical Sciences  (1.788)
  • Biochemistry and Biotechnology  (725)
  • Biochemistry  (136)
  • Nuclear reactions
Materialart
Erscheinungszeitraum
  • 1990-1994  (2.648)
Jahr
Schlagwörter
  • 1
    ISSN: 0886-1544
    Schlagwort(e): polycentric chromosome ; light microscopy ; electron microscopy ; high-pressure freezing ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-μm) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-μm wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mid anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its polefacing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 μm dia.) rods varying considerably (0.15-2.3 μm) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.
    Zusätzliches Material: 22 Ill.
    Materialart: Digitale Medien
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  • 2
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 271-272 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 3
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 4
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 199-203 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 5
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Haras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of α-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of α-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between α-actin down-regulation and stress fiber loss.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 6
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 250-263 
    ISSN: 0886-1544
    Schlagwort(e): myosin ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin-activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 7
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 309-316 
    ISSN: 0886-1544
    Schlagwort(e): digitization ; flagellum ; image analysis ; microcomputer ; simplex ; spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Methods are described for computerized analysis of digitized images obtained by scanning photomicrographs of swimming sperm flagella. After storing a series of image frames in computer memory, the entire series is analyzed automatically. For each sperm image, the sperm head is located to obtain a starting point for analysis of the flagellum. This location is obtained by minimizing image intensity along a model of the sperm head outline. The flagellum is modelled by a series of straight segments of equal length: 0.5 or 1 μm. The angles between these segments are adjusted to give minimum image intensity along the line of the model as well as minimizing smoothing functions. Extensions to analyze a series of images in each frame, and to measure the positions of beads attached to the flagellar microtubules, are also described.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 8
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 58-67 
    ISSN: 0886-1544
    Schlagwort(e): subunit composition ; Western blotting ; monoclonal antibody ; affinity-purified polyclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Sea urchin sperm outer arm dynein is a multi-subunit protein composed of heavy chains, intermediate chains, and light chains. We prepared monoclonal and affinity-purified polyclonal antibodies to heavy and intermediate chain subunits and examined whether the embryonic ciliary axonemes ofthe same species contain the polypeptides sharing epitopes with them. Ciliary axonemes contained a high molecular weight polypeptide with the exact same mobility as flagellar β-heavy chain. This polypeptide also shared epitopes with it. In contrast, no polypeptide had the exact same molecular weight as flagellar α-heavy chain and shared epitopes with it. Western blots showed that ciliary axonemes also contain three polypeptides sharing epitopes with the respective flagellar intermediate chain. The present results revealed that the α-heavy chains of flagellar and ciliary outer arm dyneins are different.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 9
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 1-6 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 10
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 121-134 
    ISSN: 0886-1544
    Schlagwort(e): clathrin ; cell-substrate adhesion ; freeze fracture ; quick-freeze ; deep-etch ; rotary- replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have used antibodies to clathrin light chains in immunocytochemical studies of acetylcholine receptor (AChR) clusters of cultured rat myotubes. Immunofluorescence and ultrastructural experiments show that clathrin is present in coated pits and in large plaques of coated membrane. Coated membrane plaques are spatially and structurally distinct from AChR-rich membrane domains and the bundles of microfilaments that are also present in AChR clusters. Clusters contain a relatively constant amount of clathrin light chain protein, which is not dependent on the amount of AChR. Clathrin plaques remain after AChR domains are disrupted by azide, or after microfilament bundles are destabilized by cytochalasin D. Extraction of myotubes with saponin removes clathrin without disrupting AChR domains. Thus, clathrin plaques, microfilament bundles, and AChR-rich domains are independently stabilized.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 11
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 33-46 
    ISSN: 0886-1544
    Schlagwort(e): dynein structure ; cilia ; development ; microtubule-based motility ; antibodies to dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Bacause of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The β-galactosidase-P72A1 fusion protein affinity-purified dynein-specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the β heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the βheavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl-terminal fragment B domain of the dynein β heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein β heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 12
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 68-79 
    ISSN: 0886-1544
    Schlagwort(e): monoclonal antibody ; centrosome ; kinetochore ; midbody ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during Gl phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 13
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 190-203 
    ISSN: 0886-1544
    Schlagwort(e): mitosis ; calcium ; diacylglycerol ; protein kinase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have treated living, intact stamen hair cells from the spiderwort plant, Tra-descantia virginiana, with 0.5 μg/ml or 60 μg/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-di-octanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to ∼8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 μg/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 μg/ml 1,2-dioctan oylglycerol in late metaphase, ∼26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 μg/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to ∼5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 μg/ml or 60 μg/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added atother times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require 〉55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 14
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 167-181 
    ISSN: 0886-1544
    Schlagwort(e): metachronal wavelength ; metachronal wave direction ; asymmetry of beating ; ciliary beating ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A mathematical model is proposed to explain the dependence of the direction and the length of the metachronal wave on parameters that characterize the ciliary beat, the dimensions of the cilia, and the geometry of their arrangement on the ciliated surface. The metach/onal wave is decomposed into two mutually perpendicular components, which are chosen in such a way that the direction of one of them is in the direction of the effective stroke. The magnitudes of the two components are determined by using the concept of the time of delay between adjacent cilia. The properties of the metachronal wave are then calculated as a function of the ciliary parameters.The results obtained with the present model predict that the direction of the wave propagation is strongly dependent on the type of metachronism in the direction of the effective stroke and the polarization in time and in space of the ciliary beat. The metachronal wavelength is found to depend on four parameters: the ciliary length, the angle of the arc projected on the cell surface by the ciliary tip during the recovery stroke, the degree of asymmetry of ciliary beat, and the portion of the cycle occupied by the pause. The metachronal wavelength is also found to be only weakly dependent on the ciliary frequency.At this stage there exists relatively little experimental information with which t o characterize fully the metachronal properties of ciliary systems. Even when only partial information exists, the model allows prediction, to within a certain range, of the direction of the wave propagation. It also suggests a possible mechanism for the influence of changes in environmental conditions on wave direction and wavelength. In severalcases in which full information does exist, good agreement between the experimental findings and the predictions of the model is found. According to this model it will be worthwhile to invest more effort in measuring the time and space polarization of ciliary beating and the times of delay between cilia.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 15
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 204-213 
    ISSN: 0886-1544
    Schlagwort(e): kinesin ; molecular structure ; immunoaffinity purification ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Previous studies with monoclonal antibodies indicate that sea urchin kinesin contains two heavy chains arranged in parallel such that their N-terminal ends fold into globular mechanochemical heads attached to a thin stalk ending in a bipartitetail [Scholey et al. 1989]. In the present, complementary study, we have used the monoclonal antikinesin. SUK4, to probe the quaternary structure of sea urchin (Strongylocentrotus purpuratus) kinesin. Kinesin prepared from sea urchin cyto-sol sedimented at 9.6 S on sucrose density gradients and consisted of 130-kd heavy chains plus an 84-kd/78 kd doublet (1 mol heavy chain: 1 mol doublet determined by gel densitomctry). Low levels of 110-kd and 90-kd polypeptides were sometimes present as well. The 84-kd/78 kd polypeptides are thought to be light chains because they were precipitated from the kinesin preparation at a stoichiometry of one mol doublet per 1 mol heavy chain using SUK4-Sepharose immunoaffinity resins. The 110-kd and 90-kd peptides, by contrast, were removed using this immunoadsorption method. SUK4-Sepharose immunoaffinity chromatography was also used to purify the 130-kd heavy chain and 84-kd/78-kd doublet (1 mol heavy chain: 1 mol doublet) directly from sea urchin egg cytosolic extracts, and from a MAP (microtubule-associated protein) fraction eluted by ATP from microtubules prepared in the presence of AMPPNP but not from microtubules prepared in ATP. The finding that sea urchin kinesin contains equi-molar quantities of heavy und light chains, together with the aforementioned data on kinesin morphology, suggests that native sea urchin kinesin is a tetramer assembled from two light chains and two heavy chains.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 16
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 17
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 18
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 251-265 
    ISSN: 0886-1544
    Schlagwort(e): Ciliary motility ; inclination ; polarity of beating ; active sliding velocity ; sliding translocation rate ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Depolarization-induced cycles of a frontal cirrus of Stylonychia were investigated by applying methods of axial-view analysis of the cilia, high-speed microcinématography, and step voltage-clamp. Rising depolarization (from 3 mV to 7ge; 30mV) increased the rate of beating from zero to maximally 58 Hz. During cyclic activity, the axis of the beat cone of a proximal segment of the cirrus was inclined by 60° (0° = perpendicular to cell surface), and was always oriented 90° counterclockwise to the power stroke. With the stimulus amplitude rising, the orientations of the power stroke and inclination were increasingly shifted in more counterclockwise directions by up to 80° After correction for inclination ( = normalization), and following planification of the track of the segment, we determined the following properties of the cycle during depolarization: The course of the cycle tended to be rounded, i.e., the ratio of major over minor amplitudes (= spatial polarity) approximated a value of 1.6 which is only two thirds of maximal spatial polarity observed during hyperpolarization. The angular velocity generally increased with rising steps of depolarization; up to +5 mV (and comparable to hyperpolarization-induced responses), the velocity maximum occurred during the return stroke. With depolarizations ≥7 mV the angular velocity maximum shifted to the power stroke so that the temporal polarity (rates of power stroke over rates of return stroke) increased from 0.4 to 1.6. Calculations of the angular velocity as referred to the proximal ciliary segment level suggest active sliding rates (between 5 and 30 nm/ms) of identified pairs of doublet microtubules. Ciliary frequency is a function of the rate of reorientation of the cyclic track; this parameter, which corresponds to the rate of translocation of active sliding between pairs of doublets, grew with the amplitude of depolarization. Translocation rates were high during transitions between the beat phases (power stroke, return stroke), and were reduced during these phases. Orientational polarograms of the mean rates of both active sliding and sliding translocation show properties of discreteness as well as continuity. The depolarization-induced changes in inclination, and the inferred patterns of sliding rate and sliding translocation rate, are compared with previous results from hyperpolarization-dependent activation of the same motor organelle.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 19
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 279-293 
    ISSN: 0886-1544
    Schlagwort(e): Cell motility ; chemotaxis ; mathematical model ; alveolar macrophages ; C5a ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Phenomenological parameters from a mathematical model of cell motility [1] are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under-agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single-cell and cell-population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single-cell level.Random motility of a cell population is characterized by the random motility coefficient, μ (analogous to a particle diffusion coefficient), whereas single-cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, χo, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single-cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single-cell parameters.The parameter μ shows a biphasic dependence on C5a concentration with a maximum of 1.9 × 10-8 cm2/sec at 10-11 M C5a and relative minima of 0.86 × 10-8 cm2/sec at 10-7 M C5a and 1.1 × 10-8 cm2/sec in the absence of C5a; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 μm/min and P varying from 22 to 32 min. χo is equal to 1.0 × 10-6 cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 μm cell length. The maximum CI measured was 0.2.Values for the population parameters μ and χo were calculated from single-cell parameter values using the aforementioned theoretical linking relationships. The values of μ and χo calculated from single-cell parameters agreed with values of μ and χo determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single-cell and cell-population parameters has not previously been reported.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 20
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 34-45 
    ISSN: 0886-1544
    Schlagwort(e): detyrosinated α-tubulin ; Drosophila embryo ; confocal microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The distribution of microtubules (MTs) enriched in detyrosinated α-tubulin (Glu-tubulin) was studied in Drosophila embryos by immunofluorescence micro-scopy by using a monoclonal antibody (ID5) which was raised against a 14-residue synthetic peptide spanning the carboxyterminal sequence of Glu-tubulin (Wehland and Weber: J. Cell Sci. 88:185-203, 1987). While all MT arrays contained tyrosinated α-tubulin (Tyr-tubulin), MTs rich in Glu-tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu-tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated α-tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu-tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for α-tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 21
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 22
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 71-74 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 23
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 87-94 
    ISSN: 0886-1544
    Schlagwort(e): benzimidazole ; anti-microtubule agents ; carbendazim ; nocodazole ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We are using molecular genetic techniques to identify sites of interaction β-tubulin with benzimidizole anti-microtubule agents. We have developed a marker-rescue technique for cloning mutant alleles of the benA, β-tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to β-tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to β-tubulin.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 24
    ISSN: 0886-1544
    Schlagwort(e): Wheat germ agglutinin ; Limax flavus agglutinin ; axonal cytoskeleton ; actin ; cytochalasin D ; axoplasmic transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Goldfish retinal ganglion cell (RGC) axons regenerating in vitro exhibit a novel mode of axoplasmic transport that entails a rapid bidirectional bulk redistribution of axoplasm, “packaged” as protruding varicosities and non-protruding phase-dense inclusions (Koenig et al.: J. Neurosci. 5:715-729, 1985; Edmonds and Koenig Brain Res. 406:288-293, 1987). We have used phase-contrast video microscopy to study transmembrane effects of surface-binding lectins on bulk transport and transport of single visible organelles in RGC axons. Our findings show that certain lectins which crosslink sialoglycoconjugates, such as wheat germ agglutinin (WGA) and the more specific sialic acid-binding lectin Limax flavus agglutinin (LFA), induce a rapid inhibition of transport activity. The LFA-induced inhibition of transport can be reversed by appropriate simple sugar haptens, and can also be antagonized by pretreatment with cytochalasin D. One of the consequences of LFA binding is an increase in RITC-conjugated phalloidin fluorescence staining of preterminal axons. The latter observation in conjunction with the antagonistic action of cytochalasin D suggests that one possible explanation for the transmembrane arrest of transport induced by crosslinking of surface sialoglycoconjugates may involve a polymerization and/or reorganization of the actin filament network which hinders translocation of mobile axoplasmic components.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 25
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 26
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 16-31 
    ISSN: 0887-3585
    Schlagwort(e): BPTI ; dithiothreitol ; DTT-sensitive mutants ; protein folding ; random mutagenesis ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 “DTT-sensitive” mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the naive wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated a most sites without completely preventing folding Because this genetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 27
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 28
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 296-297 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Zusätzliches Material: 1 Tab.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 29
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 30
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 32-40 
    ISSN: 0887-3585
    Schlagwort(e): myelin basic protein ; phosphorylation ; protein conformation ; β-structure ; thrombin ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated forms(s) of component1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phophorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in β-structure(s).
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 31
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 44-61 
    ISSN: 0887-3585
    Schlagwort(e): RBP ; RBP family ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Human serum retinol binding protein (RBP) in complex with retinol has been crystallographically refined to an R-factor of 18.1% with 2Å resolution data. The protein topology results in an anti-parallel β-barrel that encapsulates the retinol ligand. A detailed description of the protein and the binding site is provided. Our structural work has helped to define a family of proteins, many of which are carrier proteins for smaller ligand molecules. We describe the structural basis for the conservation of sequence within the family.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
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  • 32
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 82-101 
    ISSN: 0887-3585
    Schlagwort(e): chymosin ; acid proteinase ; rennin ; X-ray structure ; structure comparison ; catalytic site ; crystal packing ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The crystal structure of recombinant bovine chymosin (EC 3.4.23.4; renin), which was cloned and expressed in Escherichia coli, has been determined using X-ray data extending to 2.3 Å resolution. The crystals of the enzyme used in this study belong to the space group I222 with unit cell dimensions a = 72.7 Å, b = 80.3 Å, and c = 114.8 Å. The structure was solved by the molecular replacement method and was refined by a restrained least-squares procedure. The crystallographic R factor is 0.165 and the deviation of bond distances from ideality is 0.020 Å. The resulting model includes all 323 amino acid residues, as well as 297 water molecules. The enzyme has an irregular shape with approximate maximum dimensions of 40 × 50 × 65 Å. The secondary structure consists primarily of parallel and antiparallel β-strands with a few short α-helices. The enzyme can be subdivided into N- and C- terminal domains which are separated by a deep cleft containing the active aspartate residues Asp-34 and Asp-216. The amino acid residues and waters at the active site form an extensive hydrogen-bonded network which maintains the pseudo 2-fold symmetry of the entire structure. A comparison of recombinant chymosin with other acid proteinases reveals the high degree of structural similarity with other members of this family of proteins as well as the subtle differences which make chymosin unique. In particular, Tyr-77 of the flap region of chymosin does not hydrogen bond to Trp-42 but protrudes out in the P1 pocket forming hydrophobic interactions with Phe-119 and Leu-32. This may have important implications concerning the mechanism of substrate binding and substrate specificity.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 33
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 164-172 
    ISSN: 0887-3585
    Schlagwort(e): protein secondary structure ; sodium channel ; CD spectra analysis program ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Three scorpion toxins have been analyzed by circular dichroism in water and in 2,2,2-trifluoroethanol (TFE) solutions. These toxins were chosen because they are representative of three kinds of pharmacological activities: (1) toxin AaH IT2, an antiinsect toxin purified from the venom of Androctonus australis Hector, which is able to bind to insect nervous system preparation, (2) toxin Css II, from the venom of Centruroides suffusus suffusus, which is a β-type antimammal toxin capable of binding to mammal nervous system preparation, and (3) the toxin Ts VII from the venom of Tityus serrulatus, which is able to bind to both types of nervous systems. In order to minimize bias, CD data were analyzed by a predictive algorithm to assess secondary structure content. Among the three molecules, Ts VII presented the most unordered secondary structure in water, but it gained in ordered forms when solubilized in TFE. These results indicated that the Ts VII backbone is the most flexible, which might result in a more pronounced tendency for this toxin molecule to undergo conformational changes. This is consistent with the fact that it competes with both antiinsect and β-type antimammal toxins for the binding to the sodium channel.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 34
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 203-212 
    ISSN: 0887-3585
    Schlagwort(e): hydrogen bonding diagram ; motifs ; helical wheel ; helical net ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also be used simply to calculate the connectivities of β-strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the ψ-loop motif from the Brookhaven Data Bank.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 35
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 251-257 
    ISSN: 0887-3585
    Schlagwort(e): peroxidase ; active site ; structure conservation ; hydrophobic cluster analysis ; sequence comparison ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 36
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 237-250 
    ISSN: 0887-3585
    Schlagwort(e): elongation factor ; energy minimization ; G-protein ; Guanine nucleotide binding ; protein structure ; protein synthesis ; structural homology ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Comparative molecular modeling has been used to generate several possible structures for the G-domain of chloroplast elongation factor Tu (EF-Tuchl) based on the crystallographic data of the homologous E. coli protein. EF-Tuchl contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF-Tuchl have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation sate of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF-Tuchl have been obtained. The interactions between EF-Tuchl and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 37
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. i 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 38
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 309-314 
    ISSN: 0887-3585
    Schlagwort(e): hormone-receptor interactions ; epitope libraries ; binding selection ; fusion phage ; human growth hormone ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl-terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH-gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild-type gene III protein. The hGH-gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH-gene III phagemid particles were enriched over 5000-fold from non-hGH phage, and 8-fold from a mutant hGH phagemid following a single hGH-specific elution step from hGH receptor-coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 39
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 173-178 
    ISSN: 0887-3585
    Schlagwort(e): mitochondria ; amino acid composition ; hydrophobicity ; composition space ; membrane protein ; transmembrane region ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium. Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell. Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitrochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space. The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space. The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space. The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val. A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 40
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 213-225 
    ISSN: 0887-3585
    Schlagwort(e): molecular modeling ; energy calculations ; δ-hemolysin ; melittin ; crystal packing ; raft ; bilayer ; membrane insertion ; channel formation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Molecular modeling and energy calculations have been used to study how δ-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar “raft” structures, in which one surface is hydrophobic and the other hydrophilic. Models of δ-hemolysin crystal structure were developed using these “rafts.” These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of δ-hemolysin with six or eight monomers per channel.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 41
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 280-286 
    ISSN: 0887-3585
    Schlagwort(e): circular dichroism ; secondary structure ; insulin ; insulin analogs ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [BS9D, T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% β-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel β-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 42
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 295-304 
    ISSN: 0887-3585
    Schlagwort(e): gonadotropin-releasing hormone ; molecular dynamics ; nuclear magnetic resonance ; antagonist design ; conformation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[Δ3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-βAla], led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 43
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 334-340 
    ISSN: 0887-3585
    Schlagwort(e): protein structure ; structural comparison ; α-β barrels ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: By exhaustive structural comparisons, we have found that about one-third of the α-helix-turn-β-strand polypeptides in α-β barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the α-helix and the β-strand is somewhat constrained, and second, the β-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the α-helix. The geometry of the turn does not seem to be a major determinant of the α-β unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the α-helix and the β-strands are very long. It also occurs much less frequently in flat-sheet α-β proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which α-β barrels are constructed.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 44
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 341-351 
    ISSN: 0887-3585
    Schlagwort(e): conserved sequence ; diagnostic peptide ; superfamily classificaiton ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: From protein sequence comparison data found in the literature, a library was organized using peptide fragment sequences which are common to related proteins. Each of the fragments was then examined for its occurrence in all the protein superfamilies defined by the NBRF-PIR data base. We have selected those fragment peptides that appear exclusively in one or a few superfamilies, and thus made a library of fragment peptides that characterize specific superfamilies. Such characteristic peptides are, in general, five to seven residues long and contain unusually high proportions of glycine and cysteine. This collection is a useful resource for the classification and functional prediction of protein molecules.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 45
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 352-364 
    ISSN: 0887-3585
    Schlagwort(e): crystallography ; refinement ; structure comparison ; molecular replacement ; precision ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 Å resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 Å resolution. Nearly all of the water molecules in the well-ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe-S4 complex is slightly distorted from ideal tetrahedral coordination.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 46
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 377-385 
    ISSN: 0887-3585
    Schlagwort(e): structure deteremination ; two-dimensional NMR ; molecular mechanics ; molecular dynamics ; conformational equilibrium ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Structure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding. Ideally, one would make use of a variety of experimental observation, not just distance constraints. Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a psuedoenergy function that is minimized in a search for an optimum molecular conformation. Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis, Escherichia coli acyl carrier protein (ACP). 54 3JHNα coupling constants, 12 coupling constants for stereospecifically assigned side chain protons, and 450 NOE distance contraints were used to calculate the 3-D structure of ACP. A three-step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations. The structures calculated with the molecular mechanics approach and the molecular dynamics apporach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints. The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two-state averaging model for the protein was used, did molecular energies drop significantly.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 47
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 1-15 
    ISSN: 0887-3585
    Schlagwort(e): α-fibrous proteins ; 4-α-helix bundle ; membrane-spanning proteins ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 48
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 265-279 
    ISSN: 0887-3585
    Schlagwort(e): potential of means force ; motions of helices ; correlated fluctuations ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The concept of secondary structure motions is examined in a molecular dynamics simulation of the protein myohemerythrin. We extracted from the simulation a corresponding trajectory of helices and demonstrated that the fluctuations of the protein are dominated by a rigid shift of these secondary structure elements. The relative motions of the helices are regular, with no clear periodicity. They are bounded by ∼2 for the center of mass motions and by 20° for the relative orientations. The potential of mean force for the interactions of the helices was calculated, and the correlations between the different extended motions were investigated. It shown that the one-dimensional mean force potentials are close to quadratic for most of the helices coordinates. The anharmonicity is reflected by changes in the direction of the normal modes as a function of the energy and by the existence of multiple free energy minima for the helices packing. The multiple conformations are associated with a single type of secondary structure coordinate: the angle that describes the relative orientation of the helices in a plane perpendicular to the line connecting their center of mass.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 49
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 50
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 41-50 
    ISSN: 0886-1544
    Schlagwort(e): adhesion plaques ; cytoskeletal interactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment, was about three fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 51
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 34-40 
    ISSN: 0886-1544
    Schlagwort(e): rat liver cells ; immunoprecipitation ; immunocytochemistry ; membrane-bound proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second im-munoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-;5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunopre-cipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 52
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 23-33 
    ISSN: 0886-1544
    Schlagwort(e): centrosome ; cytaster ; MTOG ; pericentriolar material ; 51 kD protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Miniasters formed in mitotic sea urchin egg after treatment with 5% hexylene-glycol were investigated with the combined techniques of indirect immunofluo-rescence using anti-tubulin and anti-51 kD protein antibodies and electron microscopy.The formation of miniasters was dependent on the mitotic cycle. In the cytoplasm of eggs treated with hexyleneglycol at early prometaphase, a small number of microtubule fragments was observed, whereas in those treated at pro-metaphase, many miniasters and microtubule fragments were seen. When treated at metaphase, we found a great number of miniasters: 250-350 in one egg. In contrast, no miniasters were seen in eggs treated at anaphase, although many long microtubules that spread throughout the cytoplasm were observed. In the eggs treated at telophase, we scarcely noticed microtubule structures in the cytoplasm. In the center of miniasters, granules were found, showing the same size and electron density as those of the microtubule-organizing granules (MTOGs). Furthermore, the 51 kD protein, a component of the centrosome and mitotic spindle, was observed to be localized in the region of miniasters.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 53
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 71-75 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 54
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 67-70 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 55
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 99-110 
    ISSN: 0886-1544
    Schlagwort(e): ascaris ; nematode ; nematoda ; sperm ; amoeboid motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9-12. min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid-washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 μm/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move reaward together, and disappear at the junction of pseudopod and cell body. These are the termination of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55-66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 56
    ISSN: 0886-1544
    Schlagwort(e): organelle translocation ; translocator ; actin-dependent ATPase ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 57
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 139-146 
    ISSN: 0886-1544
    Schlagwort(e): organelle translocation ; cytosolic factor ; secretion ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actindependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 58
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 156-161 
    ISSN: 0886-1544
    Schlagwort(e): microtubule structure ; microtubule assembly ; electron microscopy of microtubules ; polymer stabilization ; microtubule-capping structures ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Microtubules contain in their lumens distinct structures (plugs) that influence their dynamic behavior in vitro. As observed by electron microscopy, plugs are stainoccluding structures 10-30 nm in length that occur along the lengths and at the ends of microtubules. Plugs occur at a frequency of 20-40% at the ends of microtubules assembled from cycled microtubule protein containing MAPs. While the composition of plugs is not known, preliminary evidence suggests that they are accretions of tubulin, that they are labile, and that they are more common in preparations containing MAPs. When polymers are induced to depolymerize by endwise subuit dissociation, the frequency of plugged microtubule ends increases transiently, suggesting that plugs temporarily stabilize microtubules. The functional significance of plugs may be that they prevent the sudden complete loss of microtubules through catastrophic disassembly. It is possible that plugs, by slowing the rate of disassembly, enable a polymer to add GTP-tubulin subunits, thereby forming a stabilizing GTP-cap. These observations suggest that plugs may stabilize polymers and account for the frequent transitions from shortening to growing phases that characterize dynamic instability.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 59
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 162-167 
    ISSN: 0886-1544
    Schlagwort(e): high-speed microcinematography ; photophobic response ; phototaxis ; blue light effect ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Chlamydomonas cells immobilized on micropipettes were filmed at high-speed (500 f/s) while photostimulated either by one continuous light stimulus or by pulsed light in the frequency range of cell rotation (1 or 2 Hz). Two kinds of photophobic step-up and two kinds of photophobic step-down beat frequency changes without a reversal of flagellar beat were observed after frame-by-frame evaluation of 141 records. So far, a single step-up response, the “shock” response, has been considered the only photophobic response. However, the present results show that the cells always responded to step-up as well as to step-down light stimuli. Either a decrease of beat frequency by step-up was combined with an increase by step-down (type I), or an increase by step-up was combined with a decrease by step-down (type II). Whether type I or type II was observed depended on the preirradiation of the cells. All four responses are blue-light responses with a lag-time of ∼40 ms. Nothing can be said about the photoreceptor site. Regardless, these responses cannot be the basic mechanism for phototaxis, as assumed till now, because the flagella remain synchronized during the flagella beat frequency changes. Even if they are uncoupled before and after stimulation, both flagella respond in the same sense, i.e., either both increase or both decrease their beat frequency. The behavioral relevance of these responses for Chlamydomonas is not yet clear.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 60
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 193-194 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 61
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 181-192 
    ISSN: 0886-1544
    Schlagwort(e): mitosis ; cell cycle ; microtubules ; nocodazole ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A monoclonal antibody prepared against a crude centrosome fraction from PtK cells reacts exclusively with centrosomes. Using Western blotting techniques, the antigen was identified as a protein with a molecular weight of 32 kd. Using this probe it is possible to follow pronounced shape changes of the centrosome through the cell cycle and to study its replication. When the microtubular cytoskeleton is removed by nocodazole, neither the shape nor the three-dimensional organization of the centrosome inside the cell are altered. Moreover, in spite of the cell cycle arrest caused by nocodazole, the centrosomal cycle proceeds, thus indicating its independence from the intact cytoskeleton and supporting its role as a semiautonomous organelle. On the basis of these results we hypothesize that the centrosome has two distinct functions: in the non-growing compact state during mitosis the centrosome serves as the pole organizer and during interphase it functions as the “cytocenter”.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 62
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 168-180 
    ISSN: 0886-1544
    Schlagwort(e): microtubule-associated proteins (MAPs) ; taxol ; oocyte maturation ; fertilization ; cell division ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A microtubule-binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol-dependent procedure [Vallee, 1982, J. Cell Biol. 92:435-442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS-PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration-dependent manner.Using indirect immunofluorescence and immunoelectron microscopy with the anti-70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.This 70 kD protein is the first microtubule-binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 63
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 6-10 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 64
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 11-18 
    ISSN: 0886-1544
    Schlagwort(e): intermediate filaments ; desmin ; cytoskeleton ; protein A-gold ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, inclu-ding the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 65
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 19-33 
    ISSN: 0886-1544
    Schlagwort(e): HeLa cells ; microtubule-associated proteins ; MAPIA ; vimentin filaments ; cytokeratin filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAPIA (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr.gyros: around: nemin: filament).
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 66
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 75-86 
    ISSN: 0886-1544
    Schlagwort(e): intermediate filaments ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Sea urchin spermatozoa, eggs, and embryos were labeled with the universal antibody against the intermediate filament proteins (anti-IFA) described by Pruss et al. [Cell 27:419-428, 1981] and with anti-beta-tubulin. Localization of these antibodies was by indirect immunofluorescence microscopy. Cytoskeleton of unfertilized eggs, prepared according to a procedure adapted from Kane [Exp. Cell Res. 162:495-506, 1986] or as described by Dufresne et al. [Biochem. Cell Biol. 66:780-791, 1988], and reacted with the anti-IFA demonstrate a uniformly stained background except for the nuclear areas, which appear as dark rings. During the first cell cycle, the anti-IFA staining pattern coincides with that of spindle-associated tubulin but not with the cortical pattern of microtubules. Swimming embryos reacted with the anti-IFA show a labeling located on the cilia and within the cytoplasm of each individual cell of the larva. In spermatozoa, the labeling occurs all along the flagellae. Immunoblots of proteins from eggs and embryos reveal one major protein of 117 kDa and sometimes a component of 66 kDa, both of which cosediment with tubulin during the isolation procedure of microtubules described by Vallee and Bloom [Proc. Natl. Acad. Sci. USA 80:6259-6263, 1983]. These data show that proteins homologous to the intermediate filament proteins reported in vertebrate cells are present in both gametes of sea urchins. The specific localization ofthese proteins in the spindle, the flagella, and the cilia suggest that they may play a significant role in the organization and function of the microtubular lattice of the spermatozoa and of the embryo.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 67
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 15 (1990), S. 51-62 
    ISSN: 0886-1544
    Schlagwort(e): microtubule-associated proteins ; intracellular motility ; CTPase ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules. UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium, These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 68
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 142-142 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 69
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 118-132 
    ISSN: 0886-1544
    Schlagwort(e): tubulin heterogeneity ; neural differentiation ; neuronal microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Five β-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene cβ4 and has been assigned to an isotypic family designated as Class III (βIII). In the nervous system of higher vertebrates, βIII is synthesized exclusively by neurons. A βIII-specific monoclonal antibody was used to determine when during chick embryogenesis cβ4 is expressed, the cellular localization of βIII, and the number of charge variants (isoforms) into which βIII can be resolved by isoelectric focusing. On Western blots, βIII is first detectable at stages 12-13. Thereafter, the relative abundance of βIII in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of βIII isoforms increases from one to three during neural development. This evidence indicates that βIII is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while cβ4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that cβ4 expression occurs either during or immediately following terminal mitosis, and suggests that βIII may have a unique role during early neuronal differentiation and neurite outgrowth.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 70
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 197-213 
    ISSN: 0886-1544
    Schlagwort(e): cell organelles ; MTOC ; development ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: This paper suggests that the formation and structure of the microtubular skeleton of centrioles and basal bodies can be derived from the following simple geometric principle. A closed ring of nine microtubular initiation sites defines (1) a template for the packing of 18 additional microtubular initiation sites, and (2) the shape of nine rigid arms. Upon swivelling of each arm around a point located four initiation sites away on the initial ring, the array unfolds in a manner similar to the opening of an iris diaphragm.As a consequence, the curved shape of the microtubular triplet blades arises together with the clockwise rotational sense of the slanted blades of the centriole or basal body. The final diameter of the centriole (basal body) self-adjusts. Furthermore, the pitch of the triplet blades, the taper of centrioles and basal bodies, and the change of slant of the blades towards the distal end can be derived. In addition, the model points to a method of replication of pro-centrioles (pro-basal bodies). The hypothesis was tested by the fitting of electron microscopical cross sections of centrioles of 3T3 cells to the geometric shapes predicted by the model.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 71
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 80-87 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeleton ; chemotaxis ; polymerization ; motility ; nucleation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Definition of the kinetics of ligand-activated actin polymerization in the neutrophil is important for ultimately understanding the mechanisms utilized for regulation of actin polymerization in this non-muscle cell. To better define the kinetics of formyl peptide (fMLP) -induced actin polymerization in neutrophils we determined F-actin content at 5 second intervals after activation of human neutrophils with a range (10-11-10-9M) of fMLP concentrations. The state of actin polymerization was monitored by quantifying F-actin content with NBD phallacidin binding in both flow cytometric and extraction assays. Results demonstrate three successive kinetic periods of fMLP-induced actin polymerization in neutrophils, a lag period, a 5 second period when rate of polymerization is maximal, and a period of declining rate of actin polymerization as F-actin content approaches a maximum. The duration of the lag period, the maximum rate of polymerization, and the maximum extent of polymerization all depend upon the fMLP concentration. The lag period varies from 0 to 12 seconds and is followed in 5-10 seconds by a 5 second burst of actin polymerization when the rate is as great as 9% increase in F-actin content per second. After the 5 second burst of polymerization, the rate of polymerization rapidly declines. The study defines three distinct kinetic periods of fMLP-induced actin polymerization during which important rate-limiting biochemical events occur. The mechanistic and motile implications of kinetic periods are discussed.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 72
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 16 (1990), S. 93-98 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 73
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 133-141 
    ISSN: 0886-1544
    Schlagwort(e): contractile ring ; cytoskeleton ; cell division ; cytokinesis ; zonula adhaerens ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: To examine the behaviour of the apical circumferential microfilament bundles (CMBs) associated with the zonula adhaerens (ZA)-junctions during mitosis, retinal pigment epithelial cells were labelled for F-actin, and retinas were serially sectioned for TEM. The results show that the ZA-CMB-complex persists throughout all stages of mitosis. At metaphase, the cells round up, but stay joined apically to adjacent cells by ZA-junctions. At telophase, the cleavage furrow forms asymmetrically from the basal end progressively toward the apical end, where the daughter cells remain connected by an intercellular bridge (IB). As the cleavage furrow with the contractile ring (CR) approaches the CMB, the two microfilament (MF) systems are oriented perpendicularly to each other. At the level of the CMB, the MFs of the CR connect the opposite sides of the CMB and bisect it into two CMBs, one for each of the two daughter cells. Subsequently, the CR in the IB splits into two, one on either side of the midbody. The two daughter cells, having acquired a complete CMB of their own, do not become direct neighbours, since adjacent cells, which remain joined to the apical ZA-junction of the dividing cell, are observed in the cleavage furrow, where they meet and form a ZA-junction between themselves, just below the IB. Separation of the daughter cells without losing contact with neighbouring cells at the level of the apical ZA-junction thus maintains the integrity of the epithelial sheet during mitosis.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 74
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 147-149 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 75
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 167-173 
    ISSN: 0886-1544
    Schlagwort(e): microtubule disassembly ; tubulin-binding sites ; protein concentration ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5-6 moles-mole-1 estramustine phosphate, and that the Kd of these sites is ≏ 20 μM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin-binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 76
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 150-166 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeletal dynamics ; IF depolymerization ; type III IF regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK-21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10-nm-diameter IF characteristic of interphase cells disassembled into aggregates containing short 4-6 nm filaments. During anaphase-telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 77
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 174-186 
    ISSN: 0886-1544
    Schlagwort(e): MAPs ; cold-stable microtubules ; microtubule assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5°C, induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5°C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33°C, but the microtubules depolymerized at 0°C, We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 78
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 214-226 
    ISSN: 0886-1544
    Schlagwort(e): actin ; cytoskeleton ; dynamic instability ; protozoa ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Microtubule dynamics has been studied extensively in vitro, but comparatively little information is available on the in vivo behavior of microtubules. Here we report on the assembly, disassembly, and sliding of microtubules in the giant freshwater amoeba, Reticulomyxa. We have found that treating the cell with 0.25% trypsin induces the rapid formation of exceedingly flat areas within the reticulopodial network, allowing for the direct observation of microtubule behavior by DIC optics and computer-enhanced video microscopy. In flattened areas, microtubule sliding occurs at rates of between 1 and 6.5 μm/sec. The average rate of microtubule assembly is 1.6 μm/sec, while microtubule disassembly takes place at about 4 μm/sec and can reach up to 19.5 μm/sec. We also observed many cases where a microtubule forms a hairpin loop and eventually breaks, resulting in bidirectional disassembly from the point of breakage. Our observations demonstrate sliding of cytoplasmic microtubules in vivo. The high rates of microtubule assembly/disassembly in this cell type are difficult to reconcile with conventional views of association and dissociation processes at microtubule ends and suggest unconventional mechanisms for the growth and shrinkage of microtubules.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 79
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 227-235 
    ISSN: 0886-1544
    Schlagwort(e): mitosis ; kinetochores ; cell division cycle ; protein phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Antibodies to both the C-terminal and the N-terminal regions of the 34 kd serinethreonine specific protein kinase, p34cdc2, were used to study the distribution of this protein in dividing cells and isolated chromosomes of the Indian muntjac. p34cdc2 was found to be present throughout the cytoplasm of dividing cells. In addition, a portion of cellular p34cdc2 was localized to the centrosome, kinetochore, and intercellular bridge and along kinetochore-to-pole microtubules during cell division. Tubulin-denuded metaphase kinetochores retained their association with p34cdc2. The detection of p34cdc2 within a variety of domains of the mitotic apparatus, in addition to the previous reported association with the centrosome [Bailly et al., EMBO J. 8:3985-3995, 1989; Raibowol et al., Cell 57:393-401, 1989] suggests that p34cdc2 may play a role in events associated with anaphases A and B as well as with the transition between interphase and mitosis.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 80
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 236-249 
    ISSN: 0886-1544
    Schlagwort(e): bundles ; cytomechanics ; photobleaching ; rheology ; viscoelasticity ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (〉 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 81
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 17 (1990), S. 291-300 
    ISSN: 0886-1544
    Schlagwort(e): PtK1 keratin filaments ; electrophoresis ; autoradiography ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible de-phosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 82
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 41-51 
    ISSN: 0887-3585
    Schlagwort(e): DNA binding proteins ; maximum likelihood ; CRP ; finite mixtures ; transcription regulation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Statistical methodology for the identification and characterization of protein binding sites in a set of unaligned DNA fragments is presented. Each sequence must contain at least one common site. No alignment of the sites is required. Instead, the uncertainty in the location of the sites is handled by employing the missing information principle to develop an “expectation maximization” (EM) algorithm. This approach allows for the simultaneous identification of the sites and characterization of the binding motifs. The reliability of the algorithm increases with the number of fragments, but the computations increase only linearly. The method is illustrated with an example, using known cyclic adenosine monophophate receptor protein (CRP) binding sites. The final motif is utilized in a search for undiscovered CRP binding sites.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 83
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 99-111 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 84
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 205-214 
    ISSN: 0887-3585
    Schlagwort(e): protein secondary structures ; circular dichroism ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 85
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 227-233 
    ISSN: 0887-3585
    Schlagwort(e): rhinovirus receptor ; ICAM-1 ; structure prediction ; immunoglobulin superfamily ; docking receptor to virus ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A model has been built of the amino-terminal domain of the intercellular adhesion molecule-1 (ICAM-1), the receptor for most human rhinovirus serotypes. The model was based on sequence and presumed structural homology to immunoglobulin constant domains. It fits well into the putative receptors attachment site, the canyon, on the human rhinovirus-14 (HRV14) surface in a manner consistent with most of the mutational data for ICMA-1 (Staunton, D. E., Dustin, M. L., Erickson, H. P., Springer, T. A. Cell, in press, 1989) and HRV14 (Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M. E., Murcko, M. A. Proc. Natl. Acad. Sci. U.S.A. 85: 5449-5453, 1988).
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 86
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 74-92 
    ISSN: 0887-3585
    Schlagwort(e): thermostability ; bacterial lactate dehydrogenase ; allosteric regulations ; crystallography ; molecular replacement ; coenzyme binding ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Structures have been determined of Bacillus stearothermophilus “apo” and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least-squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.Further refinement proceeded with the isomorphous holo-enzyume from Bacillus Stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.Two sulfate ions per subunit were included in the final model of the “apo” -from-one at the substrate binding site and one close to the molecular P -axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the “apo” model.This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6-bisphosphate were also analyzed.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
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  • 87
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 185-197 
    ISSN: 0887-3585
    Schlagwort(e): restriction enzyme ; DNA-protein interactions ; DNA recognition ; enzyme-substrate interaction ; active site ; site-directed mutagenesis ; protein structure-function ; DNA double-strand breaks ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The EcoRI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations. Because the EcoRI enzyme has been extremely well characterized biochemically and its structure is known at 3 Å resolution as an enzyme-DNA complex, EcoRI also serves as an important model of DNA-protein interactions. To facilitate a genetic analysis of the EcoRI enzyme, we devised an in vivo DNA scission assay based on our finding that DNA double-strand breaks induce the Escherichia coli SOS response and thereby increase β-galactosidase expression from SOS::lacZ gene fusions. By site-directed mutagenesis, 50 of 60 possible point mutations were generated at three amino acids (E144, R145, and R200) implicated in substrate recognition by the crystal structure. Although several of these mutant enzymes retain partial endonuclease activity, none are altered in substrate specificity in vivo or in vitro. These findings argue that, in addition to the hydrogen bond interactions revealed by the crystal structure, the EcoRI enzyme must make additional contacts to recognize its substrate.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 88
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 234-248 
    ISSN: 0887-3585
    Schlagwort(e): computer modelling ; Ca2+-binding proteins ; hydrophobic binding interactions ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Crystals of troponin are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecules. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 89
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 249-256 
    ISSN: 0887-3585
    Schlagwort(e): β-barrels ; β-strand ; scaffold design ; molecular modeling ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The 8-old parallel α/β-barrel topology is encountered in proteins that display an impressive variety of functions, suggesting that this topology may be a rather nonspecific and stable folding motif. Consequently, this motif can be considered as an interesting framework to design novel proteins. It has been shown that the shape of the β-sheet portion of the barrel can be approximated by a hyperboloid. This geometric object may therefore be used as a scaffold to construct an idealized eight-standard β-barrel. To facilitate the de novo design of such structures, a collection of modelling tools has been developed allowing secondary structure elements to be mapped onto the scaffold surface and rotation and translation operations to be performed about user defined axes while evaluating their contribution to the conformational energy of the system. These tools have been applied in a systematic study assessing the φ, ψ requirements to design symmetric eight standard β barrels with optimal hydrogen bonding between adjacent β-strands. It is observed that: (a) the β-sheet structure can be closed without introducing irregular stagger between β-strands and (b) the region of φ, ψ dihedral angle space compatible with the formation of regular symmetric eight standard β-barrels coincides with the φ, ψ region corresponding to average β-strands in known protein structures, suggesting that barrel closure does not impose gross constraints on β-strand geometry.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 90
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 280-290 
    ISSN: 0887-3585
    Schlagwort(e): proteases ; protein turnover ; post-translational processing ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A system has been developed or the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK-9) and the γ-subunit of nerve growth factor (nGK)-3, as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factors interactions observed in this family of proteins.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 91
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 306-316 
    ISSN: 0887-3585
    Schlagwort(e): neutral mutations ; random mutagenesis ; protein structure ; protein folding ; protein families ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A method of targeted random mutagenesis has been used in investigate the informational content of 25 residue positions in two α-helical regions of the N-terminal domain of λ repressor. Examination of the functionally allowed sequences indicates that there is a wide range in tolerance to amino acid substitution at these position. At position that are buried in the structure, there are severe limitations on the number and type of residues allowed. At most surface positions, many different residues and residue types are tolerated. However, at several surface positions there is a string preference for hydrophilic amino acids, and at one surface position proline is absolutely conserved. The results reveal the high level of degeneracy in the information that specifies a particular protein fold.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 92
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 343-357 
    ISSN: 0887-3585
    Schlagwort(e): engineered disulfide groups ; X-ray structure ; hydrophobic cavities ; water structure, altered ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The X-ray structure of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as α-helices and β-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 93
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 366-377 
    ISSN: 0887-3585
    Schlagwort(e): computer modeling ; protein ; structure ; α-carbons ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A procedure for the construction of complete protein structures from only αcarbon coordinates is described. This involves building the backbone by sequential addition of Pro, Gly, or Ala residues. This main chain structure is then refined using molecular dynamics. Side chains are constructed by sequential addition of atoms with intermediate molecular dynamics refinement. For α lytic protease (a structure that is mostly β sheet) a backbone root mean square deviation (RMSD) of 0.19 Å and an overall RMSD of 1.24 Å from the crystallographic coordinates are attained. For troponin C (67% β-helix), where the coordinates are available only for the α-carbons, a backbone RMSD of 0.41 Å and an overall RMSD of 1.68 Å are attained (fits kindly provided by Dr. Michael James and Natalie Strynadka). For flavodoxin a backbone RMSD of 0.49 Å and an overall RMSD of 1.64 Å were attained.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 94
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 378-402 
    ISSN: 0887-3585
    Schlagwort(e): conformational distance ; conformational comparison ; generalized bond matrix representation ; structure space ; evolution ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We address herein the problem of delineating the relationships between the known protein structures. In order to study this problem, methods have been developed to represent arbitrarily sized fragments of biopolymer backbone, and to compare distributions of such fragments. These methods are applied to a classification of 123 structures representing the entire set of known x-ray structures. The resulting data are analyzed (on the four-Cα length scale) to determine both the large-scale organization of the set of known structures (i.e., the relationships between large groups of structures, each comprised of proteins that are structurally related) and its local structure (i.e., the quantitative degree of similarity between any two specific structures). It is shown that the set of structures from a continuum of structural types, ranging from allhelical to all-sheet/barrel proteins. It is further demonstrated that the density of protein structures is not uniform across this continuum, but rather that structures cluster in certain regions, separated by regions of lower population. The properties of the various regions of the structural space are determined. The existence is demonstrated of strong quantitative correlations between the contents of different types of four-Cα fragments within protein structures, which imply significant constraints on the types of architecture that can occur in proteins. Analysis of the distribution of structures demonstrates some hitherto unsuspected similarities and suggests that, in some circumstances, neither structural similarity no sequence homology may be necessary conditions for evolutionary relationship between proteins. It is also suggested that these unsuspected similarities may imply similar folding mechanisms for structures of apparently different global architecture. Cases are also noted in which apparently similar structures may fold by different mechanisms. The connection between structure and dynamic properties is discussed, and a possible role of dynamics in the evolution of protein structures is suggested. The sensitivity of the methods presented herein to anomalies of structure refinement is demonstrated. It is suggested that present results provide a frame-work for analyzing experimental results on structural similarity obtained using vibrational circular dichroism spectra, which are sensitive to local backbone structure.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 95
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 30-43 
    ISSN: 0887-3585
    Schlagwort(e): molecular mechanics ; solvation energy ; trypsin ; energy minimization ; side chains ; protein crystallography ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A “knowledge-based” method of predicting the unknown structure of a protein from a homologous known structure using energetics to determine a sidechain conformation is proposed. The method consists of exchangin the residues in the known structure for the sequence of the unknown protein. Then a conformational search with molecular mechanics energy minimization is done on the exchanged residues. The lowest energy conformer is the one picked to be the predicted structure. In the structure of bovine trypsin, the importance of including a solvation energy term in the search is demonstrated for solvent accessible residues, while molecular mechanics alone is enough to correctly predict the conformation of internal residues. The correctness of the model is assessed by a volume error overlap of the predicted structure compared to the crystal structure. Finally, the structure of rat trypsin is predicted from the crystal structure of bovine trypsin. The sequences of these two proteins are 74% identical and all of the significant changes between them are on external residues. Thus, the inclusion of solvation energy in the conformational search is necessary to accurately predict the structure of the exchanged residues.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 96
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 62-81 
    ISSN: 0887-3585
    Schlagwort(e): pepsin ; aspartic proteinases ; subdomains ; structure comparison ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 Å resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 Cα pairs aligned to 0.72-0.85 Å rms for the N domains; 64-95 Cα pairs aligned to 0.78-1.03 Å rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). The subdomain is connected, or “hinged,” to a mixed β-sheet that forms one of the structurally invariant, active site ψ-loops. Relative subdomain displacements as large as a 21.0° rotation and a 5.9 Å translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 97
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 156-163 
    ISSN: 0887-3585
    Schlagwort(e): transcription activation ; secondary structure ; machine learning ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A common sequence/structural motif pattern has been identified within the steroid/thyroid hormone receptors and other transcriptional activators using a new massively parallel symbolic learning assistant computer system. The pattern appears nearly diagnostic of transcription activation, including relative activation strength, among nuclear and DNA-binding prokaryotic proteins. In cases where mutation/deletion/chimeric studies have identified the activation domain, the pattern matches within that domain. These facts and the nature of the pattern itself strongly support the idea that the patterned domain is directly involved in a protein-protein transcription activation interaction.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 98
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 195-202 
    ISSN: 0887-3585
    Schlagwort(e): simulated annealing ; computer-aided drug design ; substrate docking ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The Metropolis technique of conformation searching is combined with rapid energy evaluation using molecular affinity potentials to give an efficient procedure for docking substrates to macromolecules of known structure. The procedure works well on a number of crystallographic test systems, functionally reproducing the observed binding modes of several substrates.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 99
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990), S. 226-236 
    ISSN: 0887-3585
    Schlagwort(e): protein structure ; ionic pores ; neural membranes ; protein design ; energy minimization ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage-sensitive sodium channel and the nicotinic cholinergic receptor. We report low-energy arrangements of α-helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion-conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3-5 amphipathic α-helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar-neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area ∼ 16 Å2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of α-helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area ∼ 46 Å2). Thus, bundles of amphipathic α-helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 100
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 8 (1990) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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