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  • 1995-1999  (4,368)
  • 1995  (4,368)
  • Life and Medical Sciences  (2,263)
  • Biochemistry and Biotechnology  (1,318)
  • Engineering  (787)
  • Nuclear reactions
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  • 1995-1999  (4,368)
Year
  • 101
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 106-109 
    ISSN: 0886-1544
    Keywords: Chlamydomonas ; cilia and flagella ; protein kinase and phosphatase ; dynein-driven microtubule sliding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The following is a summary of physiological and pharmacological studies of the regulation of dynein-driven microtubule sliding in Chlamydomonas flagella. The experimental basis for the study is described, and data indicating that an axonemal cAMP-dependent protein kinase can regulate inner arm dynein activity are reviewed. In addition, preliminary data are summarized indicating that an axonemal type 1 phosphatase can also regulate dynein-drive microtubule sliding velocity. It is predicted that the protein kinase, phosphatase, and an inner dynein arm component form a regulatory complex in the axoneme.
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  • 102
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 129-132 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 1 Ill.
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  • 103
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 151-161 
    ISSN: 0886-1544
    Keywords: membrane localization ; ATPase activity ; actin binding ; calmodulin ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Although the specific functions of myosin I motors are not known, their localization to membrane structures suggests a function in membrane motility. Different myosin I isoforms in the same cell or in different cells can possess different localizations. To determine if the localization and biochemical activity of the best-characterized mammalian myosin I, chicken intestinal epithelium brush border myosin I, was dependent on determinants of the membrane or actin cytoskeleton specific to epithelial cells, we transfected the cDNA for the heavy chain of this myosin into COS cells. Transient transfection of COS cells with the chicken brush border myosin I heavy chain resulted in the production of recombinant myosin I. Recombinant brush border myosin I localized to protrusions of the plasma membrane, particularly at spreading cell edges, and also to unknown cytoplasmic structures. Some cells expressing particularly high levels of brush border myosin I possessed a highly irregular surface. Recombinant brush border myosin I purified from COS cells bound to actin filaments in an ATP-dependent manner and decorated actin filaments to form a characteristic appearance. The recombinant myosin also catalyzed calcium-sensitive, actin-activated MgATPase activity similar to that of the native enzyme. Thus, any cellular factor required for the general membrane localization or biochemical activity of brush border myosin I is present in COS cells as well as intestinal epithelium.
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  • 104
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 205-225 
    ISSN: 0886-1544
    Keywords: myofibrillogenesis ; sarcomere structure ; Z-line ; protein ruler ; actin-binding protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A 107-kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti-nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107-kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380-amino acid open reading frame composed entirely of nebulin-like 35-residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60-85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an ˜8-kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin-like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I-Z-I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin-like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin-like protein from the 600-900 kD skeletal muscle nebulin, we have named it nebulette. © 1995 Wiley-Liss, Inc.
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  • 105
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 173-186 
    ISSN: 0886-1544
    Keywords: microtubule dynamics ; microinjection ; centripetal transport ; pinocytotic vesicles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The Study of microtubule (MT) dynamics in cells has largely been restricted to events occurring over relatively short periods in nonmotile or stationary cell in culture. By using the antioxidant, Oxyrase, we have reduced the sensitivity of fluorescent MTs to photodamage and this has allowed us to image fluorescent MTs with good temporal resolution over much longer periods of time. We have used our enhanced imaging capabilities to examine MT dynamics in fibroblasts moving directionally into a wound. We found that MTs in these cells exhibited dynamic instability similar to that reported for other cells. More interestingly, we found a novel dynamic behavior of the MTs in wihch entire MTs were moved inward from the leading edge toward the cell nucleus. This centripetal transport (CT) of MTs only occurred to those MTs that were oriented with their long axis parallel to the leading edge; radially oriented MTs were not transported centripetally. Both small bundles of MTs and individual MTs were observed to undergo CT at a rate of 0.63 × 0.37 μm/min. This rate was similar to the rate of CT of latex beads applied to the cell surface and of endogenous pinocytotic vesicles in the cytoplasm. When we imaged both MTs and pinocytotic vesicles, we found that the pinocytotic vesicles were ensheathed by a small group of parallel MTs that moved centripetally in concert with the vesicles. Conversely, we found many instances of MTs moving centripetally without associated vesicles. When cells were treated with nocodazole to depolymerize MTs rapidly, the rate of pinocytotic vesicle CT was inhibited by 75%. This suggests that centripetal transport of MTs may be involved in the movement of pinocytotic vesicles in cells. In conclusion, our results show that MTs in motile cells are redistributed by a novel mechanism, CT, that does not require changes in polymer length. The centripetally transported MTs may play a role in transporting pinocytotic vesicles in the cell. © 1995 Wiley-Liss, Inc.
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  • 106
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 32 (1995), S. 318-331 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; cyclic AMP ; vinculin ; E-cadherin ; ZO-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In epithelial cells interactions between the actin cytoskeleton and cell-cell junctions regulate paracellular permeability and partcipate in morphogenesis. We have studied the relationship between supracellular morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respectively, and showed F-actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin-rich zonula adherens and in cell-substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton-insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 μM) caused actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO-1 and E-cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP. © 1995 Wiley-Liss, Inc.
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  • 107
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 132-140 
    ISSN: 0887-3585
    Keywords: protein folding ; protein stability ; mutational effects ; φ, ψ distribution ; Ramachandran map ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Changes in amino acid side chains have long been recognized to alterthe range and distribution of φ, ψ angles found in the main chain of polypeptides. Altering the range and distribution of φ, ψ angles also alters the conformational entropy of the flexible denatured state and may thus stabilize or destabilize it relative to the comparatively conformationally rigid native state. A database of 12,320 residues from 61 nonhomologous, high resolution crystal structures was examined to determine the φ, ψ conformational preferences of each of the 20 amino acids. These observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributionswere used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by -0.82 ± 0.08 kcal/mol at 20°C. Surprisingly, alanine is found to be the most ordered residue other than proline. This unexpected result is a result of the high percentage of alanines found in helical conformations. This either indicates that the observed distributions in the native state do not reflect the distributions in the denatured state, or that alanine is much more likely to adopt a helical conformation in the denatured state than residues with longer side chains. Among those residues with φ, ψ angles compatible with helix incorporation the percentage of alanines actually in helices is very similar to other residues. This and the consistent ordering of alanine relative to other residues regardless of secondary structure are evidence that φ, ψ distributions in native states reflect those in the denatured states. © 1995 Wiley-Liss, Inc.
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  • 108
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 154-167 
    ISSN: 0887-3585
    Keywords: iron-sulfur proteins ; electron transfer ; oxidation-reduction potentials ; solvent accessibility ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulations of Clostridium pasteurianum rubredoxin in the oxidized and reduced forms have been performed. Good agreement between both forms and crystal data has been obtained (rms deviation of backbone atoms of 1.06 and 1.42 Å, respectively), which was due in part to the use of explicit solvent and counterions. The reduced form exhibits an unexpected structural change: the redox site becomes much more solvent-accessible, so that water enters a channel between the surface and the site, but with little actual structural rearrangement (the rms deviation of backbone atoms between the oxidized and reduced is 0.77 Å). The increase in solvent accessibility is also seen, although to a much lesser extent, between the oxidized and reduced crystal structures of Pyrococcus furiosus rubredoxin, but no high resolution crystal or nuclear magnetic resonance solution data exist for reduced C. pasteurianum rubredoxin. The electrostatic potential at the iron site and fluctuations in the potential, which contribute to both the redox and electron transfer properties, have also been evaluated for both the oxidized and the reduced simulations. These results show that the backbone plays a significant role (62-70 kcall/mol/e) and the polar sidechains contribute relatively little (0-4 kcal/mol/e) to the absolute electrostatic potential at the iron of rubredoxin for both forms. However, both groups contribute significantly to the change in redox state by becoming more polarized and more densely packed around the redox site upon reduction. Furthermore, these results show that the solvent becomes much more polarized in the reduced form than in the oxidized form, even excluding the penetrating water. Finally, the simulation indicates that the contribution of the charged side chains to the electrostatic potential is largely canceled by that of the counterions. © 1995 Wiley-Liss, Inc.
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  • 109
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 191-192 
    ISSN: 0887-3585
    Keywords: lipopolysaccharide ; lipid A ; endotoxin ; protein structure ; acyltransferase ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of UDP-N-acetylglucosamine O-acyltransferase (lpxA) fromEscherichia coli have been obtained from solutions of sodium/potassium phosphate and dimethylsulfoxide. These crystals belong to the cubic space group P213 (a = 99.0 Å), diffract X-raysto approximately 2.5 Å resolution and contain one subunit of the enzyme in the asymmetric unit. © 1995 Wiley-Liss, Inc.
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  • 110
    ISSN: 0887-3585
    Keywords: hydrophobic moment ; peptide-cell ; membrane interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Multiple linear regression was used to quantify the dependence of the antimicrobial activity of 13 peptides upon three calculated or experimentally determined parameters: mean hydrophobicity, mean hydrophobic moment, and α-helix content. Mean hydrophobic moment is a measure of the amphiphilicity of peptides in an α-helical conformation. Antimicrobial activity was quantified as the reciprocal of the measured minimal inhibitory concentration (MIC) against Escherichia coli. One of the peptides was magainin 2, and the remainder were novel peptides designed for this study. The multiple linear regression results revealed that the amphiphilicity of the peptides was the most important factor governing anti-microbial activity compared to mean hydrophobicity orα-helix content. A better regression cf the data was obtained using In(1/MIC + constant) as the dependent variable than with either 1/MIC or In(1/MIC). These results should be useful in designing peptides with higher antimicrobial activity. © 1995 Wiley-Liss, Inc.
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  • 111
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 168-181 
    ISSN: 0887-3585
    Keywords: aspartic proteinase ; enzyme kinetics ; rule-based model ; chromogenic assay ; synthetic substrate ; inhibitor ; molecular modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to othermechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generatedto elucidate the specificity in the individual binding pockets with systematic substitutions in the P5- P2 and P2′-P3′ based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, asecond series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined forboth sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, andmouse and human renin. Important specificity-determining interactions were found in the S3 (Glu13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300)subsites. © 1995 Wiley-Liss, Inc.
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  • 112
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 113
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 193-196 
    ISSN: 0887-3585
    Keywords: urea cycle ; frog ; liver ; carbamyl phosphate synthetase ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P41212 (or its enantiomorph P43212), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (Mr of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.
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  • 114
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 199-209 
    ISSN: 0887-3585
    Keywords: chaperonins ; electron microscopy ; FTIR ; molecular modeling ; structure prediction ; contact prediction ; active site prediction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional structure of the GroES monomer and its interaction with GroEL has been predicted using a combination of prediction tools and experimental data obtained by biophysical [electron microscope (EM), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR)] and biochemical techniques. The GroES monomer, according to the prediction, is composed of eight β-strands forming a β-barrel with loose ends. In the model, β-strands 5-8 run along the outer surface of GroES, forming an antiparallel β-sheet with β4 loosely bound to one of the edges. β-strands 1-3 would then be parallel and placed in the interior of the molecule. Loops 1-3 would face the internal cavity of the GroEL-GroES complex, and together with conserved residues in loops 5 and 7, would form the active surface interacting with GroEL. © 1995 Wiley-Liss, Inc.
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  • 115
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 226-244 
    ISSN: 0887-3585
    Keywords: photosynthesis ; protein structure ; homology modeling ; molecular mechanics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three-dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. Capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. Viridis and Rb. Sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus. © 1995 Wiley-Liss, Inc.
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  • 116
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 210-225 
    ISSN: 0887-3585
    Keywords: serpin-proteinase complex ; mutants ; deamidation ; α-helix-β-sheet conversion ; homology modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The mechanism of formation and the structures of serpin-inhibitor complexes are not completely understood, despite detailed knowledge of the structures of a number of cleaved and uncleaved inhibitor, noninhibitor, and latent serpins. It has been proposed from comparison of inhibitor and noninhibitor serpins in the cleaved and uncleaved forms that insertion of strand s4A into preexisting β-sheet A is a requirement for serpin inhibitor activity. We have investigated the role of this strand in formation of serpin-proteinase complexes and in serpin inhibitor activity through homology modeling of wild type inhibitor, mutant substrate, and latent serpins, and of putative serpin-proteinase complexes. These models explain the high stability of the complexes and provide an understanding of substrate behavior in serpins with point mutations in s4A and of latency in plasmingoen activator inhibitor I. © 1995 Wiley-Liss, Inc.
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  • 117
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 259-266 
    ISSN: 0887-3585
    Keywords: homology search ; phosphodiesterases ; sequence analysis ; structure prediction ; threading ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The crystal structure of glycerol-3-phosphate cytidylyltransferase from B. subtilis (TagD) is about to be solved. Here, we report a testable structure prediction based on the identification by sequence analysis of a superfamily of functionally diverse but structurally similar nucleotide-binding enzymes. We predict that TagD is a member of this family. The most conserved region in this superfamily resembles the ATP-binding HiGH motif of class I aminoacyI-tRNA synthetases. The predicted secondary structure of cytidylyltransferase and its homologues is compatible with the α/β topography of the class I aminoacyl-tRNA synthetases. The hypothesis of similarity of fold is strengthened by sequence-structure alignment and 3D model building using the known structure of tyrosyl tRNA synthetase as template. The proposed 3D model of TagD is plausible both structurally, with a well packed hydrophobic core, and functionally, as the most conserved residues cluster around the putative nucleotide binding site. If correct, the model would imply a very ancient evolutionary link between class I tRNA synthetases and the novel cytidylyltransferase superfamily. © 1995 Wiley-Liss, Inc.
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  • 118
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 267-272 
    ISSN: 0887-3585
    Keywords: α-keratin ; intermediate filaments ; epidermal keratin ; vimentin ; keratinopathies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In intermediate filaments (IF) both epidermal keratin and vimentin molecules have been shown to have an eight residue head to-tail overlap between the rod domains of similarly directed molecules. In the case of the epidermal keratins this region has also been shown to have particular structural/functional significance since it represents a hot-spot for mutations in the four keratinopathies characterized to date. While there is good evidence that this head-to-tail overlap is present in IF containing Type III, IV, and V chains, as well as in the epidermal keratin IF (Ib/IIb), there are no data currently available for the hard α-keratin IF (Ia/IIa). Using a variety of data derived from X-ray diffraction and crosslinking studies, as well as theoretical modeling, it is now possible to demonstrate that the overlap region is not a feature of hard α-keratin IF. Indeed, it is shown that there is a nine residue gap between consecutive parallel molecules in the IF. An explanation for this observation is presented in terms of compensating disulfide bonds that occur both within the IF, and between the IF and the matrix in which the IF are embedded. © 1995 Wiley-Liss, Inc.
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  • 119
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 245-258 
    ISSN: 0887-3585
    Keywords: bacterial muramidase ; peptidoglycan ; structure comparison ; sequence motifs ; structure/function relationships ; evolutionary relationships ; X-ray structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The 70-kDa soluble lytic transglycosylase (SLT70) from Escherichia coli is a bacterial exo-muramidase that cleaves the cell wall peptidoglycan, producing 1,6-anhydro-muropeptides. The X-ray structure of SLT70 showed that one of its domains is structurally related to lysozyme, although there is no obvious similarity in amino acid sequence. To relate discrete structural features to differences in reaction mechanism and substrate/product specificity, we compared the threedimensional structure of the catalytic domain of SLT70 with the structures of three typical representatives of the lysozyme superfamily: chicken-type hen egg-white lysozyme, goosetype swan egg-white lysozyme, and phage-type lysozyme from bacteriophage T4. We find a particularly close relationship between the catalytic domain of SLT70 and goose-type lysozyme, with not only a significant similarity in overall structure, but even a weak homology in amino acid sequence. This finding supports the notion that the goose-type lysozyme takes up a central position in the lysozyme superfamily and that it is structurally closest to the lysozyme ancestors. The saccharide-binding groove is the most conserved part in the four structures, but only two residues are absolutely preserved: the “catalytic” glutamic acid and a structurally required glycine. The “catalytic” aspartate is absent in SLT70, a difference that can be related to a different mechanism of cleavage of the β-1,4-glycosidic bond. The unique composition of amino acids at the catalytic site, and the observation of a number of differences in the arrangements of secondary structure elements, define the catalytic domain of SLT70 as a novel class of lysozymes. Its fold is expected to be exemplary for other bacterial and bacteriophage muramidases with lytic transglycosylase activity. © 1995 Wiley-Liss, Inc.
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  • 120
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 22 (1995), S. 273-283 
    ISSN: 0887-3585
    Keywords: globular proteins ; protein structure analysis ; optimal rigid body superposition ; three-dimensional structural motif ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Protein structures are routinely compared by their root-mean-square deviation (RMSD) in atomic coordinates after optimal rigid body superposition. What is not so clear is the significance of different RMSD values, particularly above the customary arbitrary cutoff for obvious similarity of 2-3 Å. Our earlier work argued for an intrinsic cutoff for protein similarity that varied with the number of residues in the polypeptide chains being compared. Here we introduce a new measure, ρ, of structural similarity based on RMSD that is independent of the sizes of the molecules involved, or of any other special properties of molecules. When ρ is less than 0.4-0.5, protein structures are visually recognized to be obviously similar, but the mathematically pleasing intrinsic cutoff of ρ〉1.0 corresponds to overall similarity in folding motif at a level not usually recognized until smoothing of the polypeptide chain path makes it striking. When the structures are scaled to unit radius of gyration and equal principle moments of inertia, the comparisons are even more universal, since they are no longer obscured by differences in overall size and ellipticity. With increasing chain length, the distribution of ρ for pairs of random structures is skewed to higher values, but the value for the best 1% of the comparisons rises only slowly with the number of residues. This level is close to an intrinsic cutoff between similar and dissimilar comparisons, namely the maximal scaled ρ possible for the two structures to be more similar to each other than one is to the other's mirror image. The intrinsic cutoff is independent of the number of residues or points being compared. For proteins having fewer than 100 residues, the 1% ρ falls below the intrinsic cutoff, so that for very small proteins, geometrically significant similarity can often occur by chance. We believe these ideas will be helpful in judging success in NMR structure determination and protein folding modeling. © 1995 Wiley-Liss, Inc.
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  • 121
    ISSN: 0887-3585
    Keywords: PCB degrading enzyme ; dioxygenase ; crystallization ; polychlorinated biphenyl ; selenomethionine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.
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  • 122
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    Proteins: Structure, Function, and Genetics 22 (1995), S. 287-289 
    ISSN: 0887-3585
    Keywords: DNA repair ; PCR ; Bacillus subtilis ; herpes simplex virus ; protein-protein interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The uracil-DNA glycosylase inhibitory protein (UGI) from the bacterio-phage PBS-l has been cloned and overexpressed. The nucleotide sequence is identical to that for the previously described PBS-2 inhibitor. The recombinant PBS-l UGI inhibits the uracil-DNA glycosylase from herpes simplex virus type-l (HSV-l UDGase), and a complex between the HSV-l UDGase and PBS-l UGI has been crystallized. The crystals have unit cell dimensions a = 143.21 Å, c = 40.78 Å and are in a polar hexagonal space group. There is a single complex in the asymmetric unit with a solvent content of 62% by volume and the crystals diffract to 2.5Å on a synchrotron radiation source. © 1995 Wiley-Liss, Inc.
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  • 123
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    Proteins: Structure, Function, and Genetics 22 (1995), S. 290-292 
    ISSN: 0887-3585
    Keywords: macroH2A ; specialized nucleosomes ; fusion protein ; crystallization ; X-ray diffraction ; noncrystal-lographic symmetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Histone macroH2A has a novel hybrid structure consisting of a large nonhistone region and a region that closely resembles a full-length histone H2A. One key to understanding macroH2A function is determining the structure and function of its nonhistone region. The nonhistone region of one of the two known macroH2A subtypes was expressed in Escherichia coli and purified using affinity and molecular sieve chromatography. Crystals of the protein suitable for structural studies were grown from polyethylene glycol solutions by vapor equilibration techniques. The crystals belong to the hexagonal space group P64 (or its enantiomorph P62) with unit cell parameters: a = b = 106.2 Å, c = 125.9 Å, α = β = 90°, and γ = 120°. There are four molecules in the asymmetric unit. Self-rotation function studies revealed three twofold noncrystallographic rotation axes related approximately by 222 symmetry. These crystals have 47% solvent content and diffract to 3.8 Å resolution. © 1995 Wiley-Liss, Inc.
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  • 124
    ISSN: 0887-3585
    Keywords: aldolase ; protein complex crystallization ; crystallization screening ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P212121 with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a noncrystallo-graphic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions. © 1995 Wiley-Liss, Inc.
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  • 125
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    Proteins: Structure, Function, and Genetics 22 (1995) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 126
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    Proteins: Structure, Function, and Genetics 22 (1995), S. 73-75 
    ISSN: 0887-3585
    Keywords: thioesterase ; crystallization ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Thioesterase II from rat mammary gland has been crystallized in the presence of decanoic acid by the vapor diffusion method. The crystals belong to the orthorhombic space group P212121, and have cell dimensions, a = 52.7 Å, b = 78.0 Å, and c = 133.6 Å. The asymmetric unit likely consists of two protein monomers based on predictions from its calculated Matthews coefficient. Crystals typically diffract to at least 2.5 Å resolution and are suitable for X-ray crystallographic analysis. © 1995 Wiley-Liss, Inc.
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  • 127
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    Proteins: Structure, Function, and Genetics 22 (1995), S. 76-78 
    ISSN: 0887-3585
    Keywords: arthritis ; cartilage ; crystallization ; link protein ; proteoglycan aggregate ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cartilage extracellular matrix link protein, having molecular mass of approximately 40 kDa, is a metalloprotein that binds divalent cations and is only soluble in low ionic strength solutions. The link protein was purified from bovine trachea and has been crystallized by a vapor diffusion method using PEG 3350 as precipitant. The crystal symmetry is P1, and the unit cell dimensions are a = 43.55, b = 53.11, c = 60.10 Å, α = 90.44, β = 106.21, γ = 101.51°. The VM of 1.8 Å3/Da is consistent with the presence of two molecules of the link protein in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 Å resolution. © 1995 Wiley-Liss, Inc.
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  • 128
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    Proteins: Structure, Function, and Genetics 22 (1995), S. 100-109 
    ISSN: 0887-3585
    Keywords: protein structure ; RNA structure ; lattice model ; chain connectivity ; self-avoiding ; dynamic programming ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An algorithm based on dynamic programming gives the lattice models having the minimal RMS deviations from the actual folds of protein (RNA, etc.) chains for a given lattice and a given orientation of the macromolecule relative to the lattice. The algorithm is applicable for 3-D lattices of any kind. The accuracy of the lattice approximation increases when the distance between neighbor chain links is not rigidly fixed. Special repulsive potentials facilitate generation of self-avoiding lattice chains. The results of model building show the efficiency and precisionof this proposed general method when compared with others. © 1995 Wiley-Liss, Inc.
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  • 129
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    Proteins: Structure, Function, and Genetics 21 (1995), S. 70-73 
    ISSN: 0887-3585
    Keywords: cell cycle protein ; crystallization ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHsl crystals were grown in the presence of vanadate, a phos-phatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6122 with unit cell dimensions: a=b=94 Å, c=131.6 Å, and γ =120. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 Å. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual Vm value of 4.4 Å3/Da or a solvent content of 72%. © 1995 Wiley-Liss, Inc.
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  • 130
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    Keywords: glucocorticoid receptor ; DNA binding domain ; mutant ; yeast ; transcription factor ; transactivation ; modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Regulation of gene expression involves a large number of transcription factors with unique DNA-binding properties. Many transcription factors belong to families of related proteins that bind to similar but distinct sequences. In this study we have analyzed how amino acid substitutions at a single position in the DNA-binding domain modulate the DNA binding specificity within the nuclear receptor family of transcription factors. All possible amino acids were introduced at the first position in the DNA recognition helix, and the specificities of the mutants were analyzed using response elements containing all combinations of bases at two variable base pair positions. All mutant proteins were functional in DNA binding, and could be divided into classes of mutants with different response element specificities. By combining functional data with analysis of the structural effects of the mutations by molecular modeling, we could identify both prohibitive steric interactions as well as positive interactions, such as hydrogen bonds, that function as important determinants for specificity. Only the residues found naturally in the glucocorticoid and estrogen receptors, glycine and glutamate, produce unique binding specificities. The specificities of the other mutants overlap with each other somewhat but the substitutions clearly have potential to contribute to diversity within the nuclear receptor family. © 1995 Wiley-Liss, Inc.
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  • 131
    ISSN: 0887-3585
    Keywords: antiparallel β-sheet ; twist ; protein folding ; side chain interactions ; branched amino acids ; cystine-rich proteins ; side chain packing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cross-strand pair correlations are calculated for residue pairs in antiparallel β-sheet for two cases: pairs whose backbone atoms are hydrogen bonded together (H-bonded site) and pairs which are not (non-H-bonded site). The statistics show that this distinction is important. When glycine is located on the edge of a sheet, it shows a 3:1 preference for the H-bonded site. Thestrongest observed correlations are for pairs of disulfide-bonded cystines, many of which adopt a close-packed conformation with each cystine in a spiral conformation of opposite chirality to its partner. It is likely that these pairs are a signature for the family of small, cystine-rich proteins. Most other strong positive and negative correlations involve charged and polar residues. It appears that electrostatic compatibility is the strongest factor affecting pair correlation. Significant correlations are observed for β- and γ-branched residues inthe non-H-bonded site. An examination of the structures showsa directionality in side chain packing. There is a correlation between (1) the directionality in the packing interactions of non-H-bonded β- and γ-branched residue pairs, (2) the handedness of the observed enantiomers of chiral β-branched side chains, and (3) the handedness of the twist of β-sheet. These findings have implications for the formation of β-sheets during protein folding and the mechanism by which the sheet becomes twisted. © 1995 Wiley-Liss, Inc.
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  • 132
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 472-490 
    ISSN: 0887-3585
    Keywords: drug design ; FKBP ; FK506 ; immunophilin ; MCSS ; DLD ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An automated method for the dynamic ligand design (DLD) for a binding site of known structure is described. The method can be used for the creation of de novo ligands and for the modification of existing ligands. The binding site is saturated with atoms (sp3 carbon atoms in the present implementation) that form molecules under the influence of a potential function that joins atoms to each other with the correct stereochemistry. The resulting molecules are linked to precomputed functional group minimum energy positions in the binding site. The generalized potential function allows atoms to sample a continuous parameter space that includes the Cartesian coordinates and their occupancy and type, e.g., the method allows change of an sp3 carbon into an sp2 carbon or oxygen. A parameter space formulated in this way can then be sampled and optimized by a variety of methods. In this work, molecules are generated by use of a Monte Carlo simulated annealing algorithm. The DLD method is illustrated by its application to the binding site of FK506 binding protein (FKBP), an immunophilin. De novo ligands are designed and modification of the immunosuppressant drug FK506 are suggested. The results demonstrate that the dynamic ligand design approach can automatically construct ligands which complement both the shape and charge distribution of the binding site. © 1995 Wiley-Liss, Inc.
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  • 133
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 491-501 
    ISSN: 0887-3585
    Keywords: antibody structure ; viral neutralization ; human rhinovirus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods.1 The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known.2 The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 Å from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone3 indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding,4 might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating. © 1995 Wiley-Liss, Inc.
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  • 134
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 502-509 
    ISSN: 0887-3585
    Keywords: microtubules ; molecular motors ; electron cryomicroscopy ; decorated microtubules ; microtubule organization ; structure of microtubule/motor domain complexes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 Å periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible. © 1995 Wiley-Liss, Inc.
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  • 135
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 525-535 
    ISSN: 0887-3585
    Keywords: DNA-protein interaction ; crystal structure ; transcription factor ; gene regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Many transcription factors have an α-helix that binds to DNA bases in a specific fashion. The DNA-binding geometry of these recognition helices varies substantially. We define a set of parameters to describe the binding geometry of recognition helices and analyze specific stereochemical elements that determine particular geometries. Because the convex surface of the helix must fit into the concave surface of the DNA major groove, the number of degrees of freedom of the recognition helix is reduced from a possible six to a single angle, which we call α. The chemically interacting DNA bases and amino acid residues must lie along a common line and have the same spacing along it. This pairing of base positions with residue positions seems to restrict the binding geometry further to a set of discrete values for α. © 1995 Wiley-Liss, Inc.
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  • 136
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 510-524 
    ISSN: 0887-3585
    Keywords: lectin ; demetallized ; peptide bond isomerization ; inter-dimer interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional structure of demetallized concanavalin A has been determined at 2.5 Å resolution and refined to a crystallographic R-factor of 18%. The lectin activity of concanavalin A requires the binding of both a transition metal ion, generally Mn2+, and a Ca2+ ion in two neighboring sites in close proximity to the carbohydrate binding site. Large structural differences between the native and the metal-free lectin are observed in the metal-binding region and consequently for the residues involved in the specific binding of saccharides. The demetallization invokes a series of conformational changes in the protein backbone, apparently initiated mainly by the loss of the calcium ion. Most of the Mn2+ ligands retain their position, but the Ca2+ binding site is destroyed. The Ala207-Asp208 peptide bond, in the β-strand neighboring the metal-binding sites, undergoes a cis to trans isomerization. The cis conformation for this bond is a highly conserved feature among the leguminous lectins and is critically maintained by the Ca2+ ion in metal-bound concanavalin A. A further and major change adjacent to the isomerized bond is an expansion of the loop containing the monosaccharide ligand residues Leu99 and Tyr100. The dispersion of the ligand residues for the monosaccharide binding site (Asn14, Agr228, Asp208, Leu99, and Tyr100) in metalfree concanavalin A abolishes the lectin's ability to bind saccharides. Since the quaternary structure of legume lectins is essential to their biological role, the tetramer formation was analyzed. In the crystal (pH 5), the metal-free concanavalin A dimers associate into a tetramer that is similar to the native one, but with a drastically reduced number of inter-dimer interactions. This explains the tetramer dissociation into dimers below pH values of 6.5. © 1995 Wiley-Liss, Inc.
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  • 137
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 536-547 
    ISSN: 0887-3585
    Keywords: water ; hydrophobicity ; hydration ; X-ray crystallography ; solvation ; ordered solvent ; molecular recognition ; water-protein interactions ; drug and inhibitor design ; protein surface analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc.
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  • 138
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    Proteins: Structure, Function, and Genetics 21 (1995), S. 226-236 
    ISSN: 0887-3585
    Keywords: doeking ; Monte Carlo ; LexA repressor ; DNA binding domain ; protein-DNA interaction ; solution structure ; molecular recognition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein-DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley-Liss, Inc.
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  • 139
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    Proteins: Structure, Function, and Genetics 21 (1995), S. 256-260 
    ISSN: 0887-3585
    Keywords: α/β - barrel ; α/β - hyperboloid - 8 ; three-dimensional structure ; local tight packing of hydrophobic groups ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between Cα atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.
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  • 140
    ISSN: 0887-3585
    Keywords: carboxylate ; magnesium ; hydration ; ligand ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH3, H2S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate-Mg(II) ion is greater than 10 kcal mol-1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH3 formaldehyde, H2O, and H2S. © 1995 Wiley-Liss, Inc.
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  • 141
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    Proteins: Structure, Function, and Genetics 23 (1995), S. 97-110 
    ISSN: 0887-3585
    Keywords: bovine pancreatic trypsin inhibitor ; cluster analysis ; conformational searching ; molecular dynamics ; protein tertiary structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root-mean-square Cα deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11-18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley-Liss, Inc.
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  • 142
    ISSN: 0887-3585
    Keywords: antibody-protein complex ; influenza virus hemagglutinin ; protein recognition ; crystallization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Fab fragments from two different monoclonal antibodies (BH151 and HC45) which bind to the same antigenic region of the influenza hemagglutinin were crystallized as complexes with the hemagglutinin. The complexes crystallize in PEG 600, pH 6.0, and PEG 2000, pH 8.5, respectively. Both crystals belong to space group P321, with very similar unit cell dimensions. © 1995 Wiley-Liss, Inc.
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  • 143
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    Cell Motility and the Cytoskeleton 30 (1995), S. 1-7 
    ISSN: 0886-1544
    Keywords: actin ; cytoskeleton ; contractile ring ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cofilin is a small actin-binding protein which reguiates actin polymerization in a pH-dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis. © 1995 Wiley-Liss, Inc.
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  • 144
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    Cell Motility and the Cytoskeleton 30 (1995), S. 38-49 
    ISSN: 0886-1544
    Keywords: Listeria monocytogenes ; actin ; profilin ; DNase I ; vitamin D-binding protein ; phalloidin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail-like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthenes, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell-to-cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial-actin tail interface. We have studied the consequences of microinjecting three different actin monomer-binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11-22 m̈M) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer-sequenstering proteins, Vitamin D-binding protein (DBP; 1-2 m̈M) and DNase I (6-7 m̈M) completely block bacterial-induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10-20 min) indicating that the mechanisms by which these proteins interfere with Listeria-induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer-binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 m̈M). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria-induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer-binding proteins. © 1995 Wiley-Liss, Inc.
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  • 145
    ISSN: 0886-1544
    Keywords: microfilamentous cytoskeleton ; actin binding proteins ; actin polymerization ; annealing ; non-muscle cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Gelsolin, a Ca++ activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton-lysed PMNs, 30% of gelsolin is bound to the Triton-soluble F-actin (TSF) pool and none is bound to the Triton-insoluble F-actin (TIF) pool. Calcium-activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F-actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin-actin interactions and TIF, TSF and G-actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin-actin interactions were measured as change in the amount of EGTA-resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA-resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F-actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA-resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++ activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++ activated initial increase in TIF concurrent with a fall in TSF without a change in total F-actin or G-actin content suggest that TIF grows initially only by TSF annealing/cross-linking to TIF. Gelsolin may regulate these events. © 1995 Wiley-Liss, Inc.
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  • 146
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    Cell Motility and the Cytoskeleton 30 (1995), S. 153-163 
    ISSN: 0886-1544
    Keywords: colchicine binding site ; MTC ; cod microtubules ; bovine microtubules ; MAPs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 ± 1.5) × 105M 1at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 × 10-5 to 1 × 10-3M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10-5M colchicine no spirals were formed, while at 10-4M and 10-3M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins. © 1995 Wiley-Liss, Inc.
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  • 147
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    Cell Motility and the Cytoskeleton 30 (1995), S. 208-220 
    ISSN: 0886-1544
    Keywords: Key words: stereocilia, N-acetylated sugars, proline receptor, nematocyst discharge ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Hair bundles located on tentacles of sea anemones are morphodynamic mechanoreceptors employed to regulate discharge of nematocysts into swimming prey. Activation of chemoreceptors for N-acetylated sugars is known to induce anemone hair bundles to elongate while shifting discharge to lower frequencies matching those produced by calmly swimming prey. In the continued presence of N-acetylated sugars, activation of proline receptors is known to induce hair bundles to shorten while shifting nematocyst discharge to higher frequencies presumed to correspond to movements produced by wounded, struggling prey. In the present study, N-acetylneuraminic acid (NANA) causes stereocilia to become more intensely fluorescent in confocal optical sections of phalloidin-stained specimens, suggesting that receptors for N-acetylated sugars initate processes to increase the density of F-actin within stereocilia. Computer analysis of electron micrographs is consistent with this interpretation for large diameter stereocilia but not for small diameter stereocilia. In the continued presence of NANA, proline causes flurescence intensity of phalloidin to decrease to or below control levels. DNaseI uniformly stains large diameter stereocilia, suggesting that these stereocilia contain a pool of G-actin. Fluorescence intensity of DNaseI in stereocilia is significantly less bright in specimens exposed to NANA alone than in specimens exposed to proline in the continued presence of NANA. It appears that whereas activated receptors for NANA induce G-actin to polymerize in large diameter stereocilia, activated receptors for proline induce F-actin to depolymerize, restoring G-actin pools. © 1995 Wiley-Liss, Inc.
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  • 148
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    Cell Motility and the Cytoskeleton 30 (1995), S. 229-246 
    ISSN: 0886-1544
    Keywords: Listeria monocytogenes ; fluorescence polarization ; actin ; confocal microscopy ; mutant ; infections ; PtK2 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria-column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria-associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: (1) the surface of the bacteria, (2) the cytoplasm next to the bacteria, (3) the outer shell of the actin column, and (4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells. © 1995 Wiley-Liss, Inc.
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  • 149
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    Cell Motility and the Cytoskeleton 31 (1995), S. 1-8 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: No Abstratct.
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  • 150
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    Cell Motility and the Cytoskeleton 31 (1995), S. 34-44 
    ISSN: 0886-1544
    Keywords: microtubule ; MTOC ; mitosis ; MPM-2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In diverse cell types, monoclonal antibody MPM-2 recognizes a class of phosphorylated proteins related to microtubule organizing centers and abundant during mitosis. We have used this antibody in an attempt to identify the spatial and temporal localization of putative microtubule organizing centers in endosperm cells of the higher plant Haemanthus. Our results show that MPM-2 recognized epitope is present in interphase cells and enriched in mitotic cells. In interphase the antibody usually stains cytoplasmic granules. During the interphase-prophase transition immunoreactive material appears in the nucleus, at the nuclear envelope, and in association with microtubules. Concomitantly, we observed an increase of immunoreactivity of the cytoplasm. During mitosis the phosphorproteins recognized by MPM-2 are detected in the cytoplasm, in association with microtubules of the spindle, the phragmoplast, and in the newly-formed cell plate. After completion of mitosis, only the cell plate and cytoplasmic granules are MPM-2 positive. Extraction of the cells with Triton X-100 prior to fixation removes staining of the cytoplasm by MPM-2. The detergent resistant immunoreactive material remains associated with surrounding the nucleus microtubules of the prophase spindle, the core of kinetochore fibers, and the phragmoplast. In the phragmoplast, however, segments of microtubules which are distal to the cell plate are depleted of MPM-2.These data demonstrate that microtubule arrays of endosperm cells are phosphorylated during mitosis. Thus, similar to animal cells, interphase and mitotic microtubules of higher plants have different properties. Additionally, the localization of detergent resistant MPM-2 antigen points to the difference in microtubule nucleation/organization between higher plant and animal cells.
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  • 151
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    Cell Motility and the Cytoskeleton 31 (1995), S. 59-65 
    ISSN: 0886-1544
    Keywords: flagella ; cane-shaped bend ; principal bend ; calcium ; membrane depolarization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To investigate the mechanism of the flagellar quiescence in sperm, we examined the effect of electric stimulation of individual spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Stimulation with a suction electrode attached to the sperm head elicited a flagellar quiescence response, in which the sperm showed a typical cane-shaped bend in the proximal region of the flagellum when the electrode was used as anode. Cathodic stimulation also induced quiescence, but was much less effective than anodic stimulation. During the quiescence response, which lasted for 1-3 s, no new bend was initiated, and subsequently the flagellum resumed normal beating. The quiescence response required the presence of Ca2+ (〉2 mM) in sea water, and was inhibited by Co2+ and La3+. At low Ca2+ concentrations (2-5 mM), the angle of the cane-shaped bend was smaller than that at 10 mM Ca2+; thus the angle of the cane-shaped bend, characteristic of the quiescence response is dependent on Ca2+ concentration. These results suggest membrane, followed by an influx of Ca2+ into the flagellum through Ca2+ channels. The increase in Ca2+ concentration within the flagellum affects the amount of sliding and thus produces a cane-shaped proximal bend of various angles, white inhibiting both the propagation of the proximal bend (principal bend) and the formation of a new reverse bend.
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  • 152
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    Keywords: cleavage furrow ; cytokinesis ; contractile ring ; microfilament ; stress fibers ; microfilament networks ; intestinal epithelium ; spleen cells ; dorsal root ganglia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithellal, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like -structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.
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  • 153
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    Cell Motility and the Cytoskeleton 31 (1995), S. 140-146 
    ISSN: 0886-1544
    Keywords: cAMP ; ATP ; hypoxia ; motility initiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Bovine sperm that were subjected to extended anoxia (2.5 h) in the absence of glycolytic substrates then diluted into oxygenated medium were immotile but metabolically active, producing ATP from lactate via oxidative phosphorylation. In response to anoxia sperm ATP titers dropped from 15-20 μmoles/108 cells to 1-2 μmoles/108 cells in the first 5 min then remained extremely low until reoxygenation. Cyclic AMP titers declined slowly over the anoxic period, but did not show the same scale of depression as ATP. After dilution and re-oxygenation ATP recovered to pre-anoxia levels within 1 min, and cAMP rose to about the pre-anoxia levels. However, motility, which varied quantitatively and qualitatively between ejaculates prior to anoxic treatment, was substantially depressed after extended anoxia in all cases; progressive motility was almost non-existent in post-anoxic sperm. Addition of isobutylmethylxanthine or Cibacron Blue F3GA, both putative phosphodiesterase inhibitors, stimulated a transient peak of cAMP, which was accompanied by motility stimulation. These techniques provide a protocol to manipulate and dissect the biochemical pathways of motility initiation in mammalian sperm.
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  • 154
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    Cell Motility and the Cytoskeleton 31 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 155
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    Cell Motility and the Cytoskeleton 31 (1995), S. 225-240 
    ISSN: 0886-1544
    Keywords: cell-substratum adhesion ; lamellar contractility ; locomotion ; silicone rubber ; traction forces ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A means of determining quantitative maps of the tractions exerted by locomoting cells on a substratum has been developed. This method is similar to the Harris silicone substratum assay [Harris et al., 1980: Science 208:177-179], but uses an improved non-wrinkling film that deforms more predictably in response to traction forces. The method also utilizes a mathematical analysis of rubber deformation to produce the final map of the distribution of tractions. The resulting maps consistently showed that fish keratocytes exert a steady-state “pinching” on the substratum, perpendicular to the cell's direction of locomotion. No significant rearward tractions were detected at or near the front edge of the cell. Likewise, no significant forward tractions associated with peeling of adhesions were found at the back of the cell. A second assay uses deflection of a lightly attached glass microneedle to measure the total force exerted by locomoting cells. Forces of approximately 4.5 × 10-3 dyn were required to “stall” locomoting keratocytes. The implications of these findings for cell movement are discussed.
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  • 156
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    Cell Motility and the Cytoskeleton 31 (1995), S. 255-258 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 157
    ISSN: 0886-1544
    Keywords: neurofilament ; phosphorylation ; cdk5 ; cdc2 ; cyclin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for in the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recoginizing in vivo phosphorylation sites (SM131, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract. © 1995 Wiley-Liss, Inc.
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  • 158
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    Keywords: thymosin β4 ; actin ; stress fibers ; cleavage furrows ; cytokinesis ; cell spreading ; PtK2 cells ; microinjection ; transfection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Thymosin β4 (Tβ4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of incresing Tβ4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of Tβ4 at a concentration (approximately 28 μM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular Tβ4 concentrations were increased by three different methods: (1) microinjection of an RSV vector containing a cDNA for Tβ4; (2) transfection with the same vector; and (3) microinjection of purified Tβ4 protein. The plasmid coding for Tβ4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure Tβ4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, Similar microinjection of pure thymosin β4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow re-gressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these Tβ4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. I 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 160
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    Cell Motility and the Cytoskeleton 32 (1995), S. 103-105 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 161
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    Cell Motility and the Cytoskeleton 32 (1995), S. 121-124 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 162
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    Cell Motility and the Cytoskeleton 32 (1995), S. 133-135 
    ISSN: 0886-1544
    Keywords: cytoplasmic dynein ; motor domain ; mutational analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The highly conserved lysine residue in the putative hydrolytic ATP-binding motif of the yeast cytoplasmic dynein heavy chain was replaced with leucine. The mutation was generated by a two-stage transformation method designed for genomic site-directed mutagenesis. Preliminary observations show that the effects of this alteration on the cellular roles of dynein are indistinguishable from those of a disruption mutation in which the entire motor domain is not expressed. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 164
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    Cell Motility and the Cytoskeleton 32 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 165
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    Cell Motility and the Cytoskeleton 32 (1995), S. 299-304 
    ISSN: 0886-1544
    Keywords: 3T3 cells ; CV1 cells ; cell motility ; infrared ; photobiology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: 3T3 mouse fibroblasts responded differently to specific near-infered signals than epithelial CV1 cell. Furthermore, signals with the same wavelength and energy changed the percentages of attracted and repelled 3T3 cells if their intensity modulation was altered. I found this result in a 22 month long study which established a spectrum of motile responses of 781 individual 3T3 cells and 148 CV1 cells to the near-infrared emissions of microscopic, pulsating light sources using the infrared spot-irradiation phase-contrast (IRSIP) microscopic [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. Thus the response of cultured, mammalian cells to near-infrared light signals is not merely a matter of total energy absorption by cirtain cytoplasmic componets. Since it seems to depend on the cell type and the temporal pattern in which the light energy is emitted, it appears to imply the existence of a new kind of cellular information. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 37-45 
    ISSN: 0886-1544
    Keywords: Entamoeba histolytica ; adhesion plates ; cytoskeleton ; fibronectin binding and degradation ; signaling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Entamoeba histolytica trophozoites are pleiomorphic and highly motile cells. Although scarce fibrous material can be identified in the cytoplasm as elements of an organized cytoskeleton, clearly defined actin-containing structures are formed at the sites of cell-matrix contact upon the interaction of trophozoites with fibronectin (FN) and other extracellular matrix substrates. These structures are reminiscent of the adhesion plaques or focal contacts found in higher eukaryotic cells, where actin filament bundles insert into specialized regions of the plasma membrane and function as signal transduction organelles. Thus, the formation of adhesion plates in this parasitic ameba could be related to specific signaling responses involved in its invasive behavior. Here, we report the isolation of amebic adhesion plates and the results of their structural and molecular analyses. Filaments, with the characteristic diameter of F-actin, radiating from an electrondense matrix, are the main feature. Actin is one of the main protein components of the plate; other proteins identified are a FN-binding protein - previously reported as a “putative” FN receptor - the actin-binding proteins myosin II, myosin I, α-actinin, vinculin, and tropomyosin. The presence in the isolated plates of several proteases and protein kinases, in particular pp125FAK, is also demonstrated. Our results suggest that adhesion plates in amebas are dynamic membrane-cytoskeletal complexes participating not only in the attachment to FN substrates but also providing the structural basis for their involvement in parasite locomotion and invasiveness. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 46-54 
    ISSN: 0886-1544
    Keywords: sliding disintegration ; Tetrahymena ; active site ; ribose-modified ATP ; dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Axonemal sliding involves both sliding velocity and the extent of sliding, that is how many doublets slide. It is clear that axonemes cannot beat if all doublets were to slide simultaneously, thus sliding extent is important. Using the turbidimetric assay of sliding disintegration of Tetrahymena axonemes, we examined the sliding extent and the effect of ADP, ATP, and ATP analogs on the sliding extent. Of course, ATP is necessary to produce sliding disintegration, but ATP alone did not produce extensive sliding disintegration. The addition of ADP allowed greater extent of sliding disintegration. The additions of higher ATP concentration even in the presence of ADP inhibited sliding disintegration. We also observed sliding disintegration using ribose-modified ATP analogs, anthraniloylATP, and methylanthraniloylATP. The extent of sliding disintegration was proportional to the analog concentration. Thus in contrast to ATP, higher analog concentration was not inhibitory. These results indicate that high ATP concentration acts to inhibit the extent of sliding disintegration and that ADP relieves this inhibition. We propose a model in which the affinity of multiple cooperative active sites are regulated by binding of ATP or ADP to a regulatory site. This model provides a mechanism by which nucleotides regulate the extent of sliding necessary for effective axonemal bending. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 90-94 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Cell Motility and the Cytoskeleton 32 (1995), S. 98-102 
    ISSN: 0886-1544
    Keywords: dynein ; mutants ; in vitro motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chlamydomonas flagella contain as many as 11 different dynein heavy chains, three in the outer arm and eight in the inner. Several lines of evidence suggest that these different dyneins are functionally diverse. This diversity may be important for the generation of axonemal undulating movement.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 125-128 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 171
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    Cell Motility and the Cytoskeleton 32 (1995), S. 136-144 
    ISSN: 0886-1544
    Keywords: DYH1B ; dynein family ; motor proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Analysis of sequence relationships in dynein heavy chains shows that dynein motor proteins comprise a single homologous family with three main branches, cytoplasmic dynein, axonemal dynein, and a third branch represented by DYH1B that lies between the other two. In all branches of the family the dynein heavy chain has four copies of the P-loop motif for a nucleotide-binding site spaced ∼300 residues apart in its midregion, with the amino acid sequence GPAGTGKT in the P-loop of the hydrolytic ATP-binding site. Cytoplasmic dyneins appear more primitive in that the heavy chain usually occurs as a homodimer, with traces of the early evolution of its four P-loop motifs by gene duplication being recognizable. In the axonemal subfamily the heavy chains occur as heterodimers or heterotrimers encoded by multiple genes, and their non-hydrolytic P-loop motifs are much more divergent with little trace of their origin by gene duplication. The DYH1B subfamily is more closely related to the cytoplasmic dyneins in sequence, but appears related to axonemal dyneins in function since it becomes upregulated during reciliation and has not been found in organisms, such as yeast and Dictyostelium, that are totally without cilia or flagella.
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  • 172
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    Cell Motility and the Cytoskeleton 32 (1995), S. 163-172 
    ISSN: 0886-1544
    Keywords: actin ; C-terminus ; α-actinin ; myosin ; myofibrils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A series of deletions was made from the C-terminal end of actin by inserting termination codons into a full length cDNA of human α-skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNA were translated in vitro using 35S-labeled methionine. The 35S-labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S-1, bind a 27 kDa proteolytic fragment of α-actinin, and incorporate into myofibrils in vitro. Removal of the C-terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S-1 or the 27 kDa fragment of α-actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356-365 is due to the important role Trp356 plays in maintaining hydrophobic bonds between three non-contiguous segments of actin. We also suggest that removal of residues 366-372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibrils. © 1995 Wiley-Liss. Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 226-232 
    ISSN: 0886-1544
    Keywords: Z-line interconnections ; honey-bee flight muscle ; transverse cytoskeletal network ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Located at the level of the Z-line, the transverse cytoskeletal network of insectflight muscle interconnects adjacent myofibrils with one another, and interconnects peripheral myofibrils with the cell membrane. This network has been presumed to keep myofibrils in register, or to distribute tension laterally among myofibrils. In this study, we used scanning-electron microscopy to reveal details of the three-dimensional arrangement of this network. The network is seen to interconnect longitudinal elements of the cytoskeletal network which surround each myofibril. The arrangement is not unlike that seen in vertebrate skeletal muscle. Interestingly, the transverse network makes contact with cell components such as dense bodies and mitochondria. Such contacts imply potential roles over and above those noted above. The network may be involved not only in mechanical function, but possibly also in intracellular communication. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 273-288 
    ISSN: 0886-1544
    Keywords: microtubules ; γ-tubulin ; polarized epithelia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubules oriented in the apicobasal axis of columnar epithelial cells arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF-B (hepatocyte), Caco-2 (intestine), and Madin-Darby canine kidney (MDCK). Caco-2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole-induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF-B and Caco-2 cells contained two pools of γ-tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non-centrosomal γ-tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal γ-tubulin in apical cytoplasm suggests multiple mechanisms by which microtubule nucleation might occur in epithelial cells. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Cell Motility and the Cytoskeleton 30 (1995), S. 26-37 
    ISSN: 0886-1544
    Keywords: myosin ; myosin-I ; unconventional myosin ; brush border ; epithelia ; membrane ; phospholipid ; fluorescence microscopy ; actin ; calmodulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Brush border myosin-I (BBMI) is associated with the membrane of intestinal epithelial cells where it probably plays a structural role. BBMI also has been identified on Golgi-derived vesicles in intestinal epithelial cells where it may translocate vesicles into the brush border. However, the mechanochemical activity of BBMI bound to a phospholipid membrane has not been described. This study reports that phospholipid membrane-associated BBMI displays ATPase activity when bound to phospholipids, but does not move actin filaments when associated with a phospholipid bilayer. BBMI does not bind significantly to brush border membrane lipids, which contain about 16% phosphatidylserine (PS), in either a pelleting or planar membrane assay. Similarly, planar membranes containing 20% PS do not bind a significant amount of BBMI. Increasing the concentration of PS to 40% does result in the binding of BBMI to both vesicles and planar membranes. This binding is enhanced with increased Ca2+ concentrations. BBMI retains its ATPase activity when bound to phospholipid vesicles containing 40% PS. However, BBMI attached to a phospholipid bilayer surface does not move actin filaments, even though the amount of BBMI bound to the lipid surface, as reflected by the number of actin filaments associated with bilayer-bound BBMI, is sufficient to observe motility in control experiments. When membrane fluidity is reduced by adding cholesterol to the membrane lipids containing 40% PS, BBMI still binds to the membrane, but again no actin filament motility is observed. The lack of binding by BBMI to brush border membrane lipids and the absence of membrane-associated BBMI mechanical activity suggest that factors in addition to membrane lipids are necessary for membrane-associated myosin-I motility. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 30 (1995), S. 171-182 
    ISSN: 0886-1544
    Keywords: tubulin ; post-translational modification ; glutamylation ; tyrosination ; dipeptide antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two monoclonal antibodies, GLU-1 and A1.6, raised against γ-L-glutamyl-L-glutamic acid dipeptide (Glu-Glu) and Ca2+ -dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu-Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross-reaction of the antibodies to the α-tubulin subunit. α-Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and A1.6 specifically recognize the detyrosinated from of α-tubulin. When microtubule protein purified from brain was probed, not only α-but also, to a lesser extent, β-tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the γ position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class IIIβ isotype at amino acid position 437 to 439, it is suggested that GLU-1 and A1.6 are able to recognize the glutamylated form of β-tubulin. These results indicate that the C-terminal Glu-Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of α-tubulin and the glutamyl side chain of β-tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post-translationally modified tubulin subunits. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 30 (1995), S. 252-260 
    ISSN: 0886-1544
    Keywords: axoneme ; ciliary regulation ; cyclic nucleotides ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca2+ -unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca2+ -dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells (“models”) were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated “on blot” by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells. Ciliary p48 may be part of the mechanism that regulates the orientation of the ciliary power stroke. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 31 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 30 (1995), S. 301-309 
    ISSN: 0886-1544
    Keywords: MAP5 ; high-molecular weight MAPs ; tubulin ; actin ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion-exchange chromatographic steps and results in 〉95% pure MAP1B with a typical recovery of about 25-30 mg/kg of brain tissue. SDS-PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti-MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co-sediments with taxol-stabilised microtubules. Co-incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G-actin nor F-actin nor co-sediments with F-actin, suggesting that it is not an actin-binding protein.
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    Cell Motility and the Cytoskeleton 31 (1995), S. 22-33 
    ISSN: 0886-1544
    Keywords: amphibian ; axonemes ; cilia ; dynein ; lung ; respiratory ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes 〉95% of the outer arm dynein (OAD), and that the beat frequency of OAD-depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199-209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The presen study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released ∼ 1.4 × 107 axonemes during isolation, yielding ∼ 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring ∼ 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0-8 M urea, 3-5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV-induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (Mr 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent Mr of the dynein heavy chains was determined to lie between 430-420 kD. Eight discrete polypeptides (putative intermediate chains, IC1-IC8, Mr 175-56 kD) copurified with the α- and β-heavy chains by microtubule-alloaffinity.Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two-headed particle represents the entire newt respiratory outer arm dynein.
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    Cell Motility and the Cytoskeleton 31 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 31 (1995), S. 130-139 
    ISSN: 0886-1544
    Keywords: dynein ; flagella ; Chlamydomonas mutants ; viscosity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The propulsive force generated by Chlamydomonas mutants deficient in flagellar dynein was estimated from their swimming velocities in viscous media. The force produced by wild-type cell increased by 30-40% when viscosity was raised from 0.9 to 2 cP but decreased as viscosity was further raised above 6 cP. The biphasic dependence of force generation on viscosity was also observed in the mutant idal, which lacks the II component of the inner-arm dynein. The mutant ida4, which lacks the inner-arm 12 component, was extremely susceptible to viscosity and stopped swimming at 6 cP, at which other mutants could swim. In contrast, odal, which lacks the entire dynein outer arm, produced a fairly constant force of about one-third of the wild-type value, over a viscosity range of 0.9-11 cP. In demembranated and reactivated cell models of the wild type, the propulsive force decreased monotonically as viscosity increased. Thus the increase in force generation at about 2 cP observed in live cells may be caused by some unknown mechanism that is lost in cell models. The cell models of odal, in contrast, did not show a marked change in force generation with the change in viscosity. These results indicate that the force generation by the outer-arm dynein greatly depends on viscosity or the velocity of movement, whereas the complete set of inner-arm dynein present in the odal axoneme produces a fairly constant force at different viscosities. These different properties of inner and outer dynein arms should be important in the mechanism that produces flagellar beating.
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    Cell Motility and the Cytoskeleton 31 (1995), S. 113-129 
    ISSN: 0886-1544
    Keywords: Arabidopsis ; centrosome ; CIPC ; colchicine ; cytokinesis ; γ-tubulin ; microtubule ; mitosis ; phragmoplast ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: γ-Tubulin-specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner. During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate. γ-Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers. At higher drug concentrations, γ-tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast. During UV-induced recovery from colchicine, γ-tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei. With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles. In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal. As with CIPC, γ-tubulin is concentrated at focal arrays. Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts. These results support a preferential association between γ-tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts. Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but γ-tubulin may also serve another function, such as in structural stabilization.
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    Cell Motility and the Cytoskeleton 31 (1995), S. 177-195 
    ISSN: 0886-1544
    Keywords: focal adhesion ; stress fiber ; vinculin ; talin ; integrin ; focal adhesion kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Human fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti-α-actinin, and anti-myosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types f stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron-dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber-membrane association site by using immunocy-tochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including β1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti-integrin αv subunit or anti-focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber-membrane contact has some important functions different from those of the focal adhesion.
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  • 188
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    Cell Motility and the Cytoskeleton 31 (1995), S. 215-224 
    ISSN: 0886-1544
    Keywords: RBL-2H3 cells ; vinculin ; mast cells ; talin ; cytoskeleton ; permeabilized ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Adherence of cells to the extracellular matrix via focal adhesions is known to modulate many cellular functions. However, the role of focal adhesions in the regulation of secretion is unclear. To examine this we have used the RBL-2H3 rat mast cell line, in which we and others have observed cytoskeletal rearrangements and increased cell spreading during secretion. All activators of secretion examined, whether acting specifically through or bypassing the IgE-receptor, induced the assembly of focal adhesions, as defined by the localization of vinculin and talin. The extent of focal adhesion formation correlated with the extent of secretion and the time course of secretion also correlated with that of the assembly of focal adhesions. To examine the mechanism by which focal adhesion formation occurred, the protein kinase C inhibitor bisindolylmaleimide was used. Bisin-dolylmaleimide caused complete inhibition of both secretion and focal adhesion formation induced by antigen or the calcium ionophore A23187. Although PMA did not induce secretion, it induced focal adhesion assembly which was inhibited by bisindolylmaleimide. The inhibitor had no effect on secretion or focal adhesion formation induced by the ATP analogue, ATPγS in permeabilized cells, indicating ATPγS acts after the activation of protein kinase C in the secretory pathway. These data provide novel evidence that the formation of focal adhesions may have a role in the process of secretion from mast cells.
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  • 189
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    Cell Motility and the Cytoskeleton 31 (1995), S. 259-272 
    ISSN: 0886-1544
    Keywords: microtubules ; transfection ; hemagglutinin antigen ; autoregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A Chinese hamster β-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HAβ1-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HAβ1-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HAβ1-tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HAβ1-tubulin. The synthesis of both endogenous β-tubulin and HAβ1-tubulin was repressed by colchicine. The HAβ1-tubulin incorporated into microtubules to the same extent as the endogenous β-tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, trasfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HAβ1-tubulin reduced the synthesis of endogenous wild-type β-tubulin but increased the synthesis of α-tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of α-tubulin was found. The results indicate that expression of excess exogenous β-tubulin perturbs the synthesis of endogenous α-tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of α- and β-tubulin for assembly. © 1995 Wiley-Liss, Inc.
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  • 190
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    Cell Motility and the Cytoskeleton 32 (1995) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 191
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    Cell Motility and the Cytoskeleton 32 (1995), S. 10-25 
    ISSN: 0886-1544
    Keywords: desmosomes ; embryonal carcinoma ; epithelia ; intermediate filaments ; keratins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytoskeletal intermediate filaments (IFs) constitute a diverse family of proteins whose members are expressed in tissue-specific patterns. Although vimentin IFs are normally restricted to mesenchyme, a variety of cell types express vimentin alone or together with cell-specific IFs during growth, differentiation, and neoplasia. In this study, we have investigated the influence of increased vimentin expression on the simple epithelial cell phenotype. An expression vector encoding a human vimentin cDNA was transfected into the murine HR9 endoderm and F9 embryonal carcinoma cell lines, which serve as models for early extraembryonic epithelial differentiation. Stable clones that expressed varying levels of the human vimentin were characterized by immunofluorescence and biochemical analysis. A relatively high level of vimentin expression in HR9 and differentiated F9 epithelial cells resulted in aberrant vimentin structures with a co-collapss of keratin K8/K18 filaments and lowered amounts of keratin protein. In F9 epithelial cells, the desmosomal proteins DP I/II did not appear to localize to cell surface desmosomes but rather co-aggregated with the perturbed IFs. Although overall cell morphology was not dramatically altered, individual nuclei were distorted by excess intracellular vimentin. Furthermore, cell proliferation as well as the cell spreading response time were slowed. There appears to be a threshold effect regarding overall vimentin levels as cells that expressed lower amounts of the human vimentin exhibited no obvious structural nor biological effects. Our results demonstrate that wild-type vimentin can act as a “mutant” protein when present at high intracellular levels, inducing a variety of phenotypic changes. © 1995 Wiley-Liss, Inc.
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  • 192
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    Cell Motility and the Cytoskeleton 32 (1995), S. 55-64 
    ISSN: 0886-1544
    Keywords: retina ; photoreceptor cells ; cytoskeleton ; centrin ; Ca2+-binding proteins ; mammals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Photoreceptor cells of vertebrate retinae are highly specialized ciliary cells. Their non-motile ciliated structure is restricted to the so-called connecting cilium at the joint between the light sensitive outer segment and the metabolically active inner segment. Extensive bidirectional intracellular transport between both segments is forced to occur through this tight connecting cilium. In the present study it is shown that the Ca2+-binding, phospho-protein centrin is present in mammalian retinae. Western blot and immunoprecipitation experiments reveal that anti-centrin antibodies react with purified photoreceptor cell fractions of retinae in bands at a molecular weight of 20 kDa, the molecular weight of centrins found in other cells. Indirect immunofluorescence analysis of cryosections through retinae of different mammalian species show that centrin is present only in centrosomes and basal bodies but also more extensively at the linkage between the inner and the outer segment of the photoreceptor cells. Immunocytological studies on isolated rod cells and immunoelectron microscopy clearly demonstrate a unique presence of centrin in the connecting cilium of photoreceptor cells. High molecular identity between centrins in lower eukaryotes and mammals indicates that centrin may play a role in cellular motility and/or in microtubule severing in the mammalian retina. © 1995 Wiley-Liss, Inc.
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  • 193
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    Cell Motility and the Cytoskeleton 32 (1995), S. 65-79 
    ISSN: 0886-1544
    Keywords: Ciona ; flagella ; motility ; tyrosine kinase ; cAMP-dependent kinase ; spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A specific peptide inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKI-peptide) is a very effective inhibitor of the cAMP-dependent activation of motility of Ciona spermatozoa, when the PKI-peptide is present at the beginning of incubation of demembranated spermatozoa with cAMP and ATP. Under conditions where approximately 120 sec is required for full activation of motility, the window of sensitivity to the PKI-peptide lasts for only 25-30 sec. Examination of sperm pellet proteins labeled with 32P ATP during activation reveals a major 25 kDa phosphoprotein and 2 minor phosphoproteins whose phosphorylation is highly sensitive to inhibition by the PKI-peptide and essentially complete during this early phase. These sperm proteins appear to be immediate substrates for cAMP-dependent protein kinase, and phosphorylation of one or more of these appears to be required, but not sufficient, for activation of motility. The phosphorylation of other proteins is reduced or eliminated when PKI-peptide is present at the beginning of incubation, but is unaffected by later addition of PKI-peptide. Some of these substrates appear to be likely candidates for axonemal proteins that must be phosphorylated during the later stages of incubation in order to complete the activation process. This selection is based upon a high degree of inhibition by inclusion of PKI-peptide or other inhibitors at the start of the incubation process, on near-completion of their phosphorylation by the end of the 2 min incubation period required for activation of motility, and evidence that these proteins are phosphorylated during in vivo activation of motility. Although these observations suggest the presence of a second kinase activity that is upregulated by the initial activation of the cAMP-dependent protein kinase, assays using exogenous substrates have not yet been able to identify such a kinase activity. © 1995 Wiley-Liss, Inc.
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  • 194
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    Cell Motility and the Cytoskeleton 32 (1995), S. 110-113 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 195
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    Cell Motility and the Cytoskeleton 32 (1995), S. 114-120 
    ISSN: 0886-1544
    Keywords: cilia ; dynein arm activity ; axonemal structure ; hydrodynamics ; computer modelling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dominance of viscous forces in the generation of propulsive thrust by cilia is emphasised. Fourier analysis indicates that ciliary bends consist of circular arcs joined by linear segments; this arc-line shape appears to be a property associated with the molecular mechanism responsible for bending the cilium and is unchanged by variations in the external viscous loading on the organelle. The flexibility of a computer-generated model of axonemal structure is demonstrated by the incorporation of recent data concerning the surface lattice of the microtubules. Computer simulations using the model show that predictions based on stochastic, rather than co-ordinated, dynein arm activity provide a qualitative match to experimental observations of microtubules gliding over fields of dynein molecules.
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  • 196
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    Cell Motility and the Cytoskeleton 32 (1995), S. 162-162 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 197
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    Cell Motility and the Cytoskeleton 32 (1995), S. 145-150 
    ISSN: 0886-1544
    Keywords: filament translation ; insect-flight muscle ; rigor-stretch model ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To investigate the pattern of actin-filament translation in the intact myofibrillar matrix, we carried out electron micrographic experiments on the “rigor-stretch” model of insect-flight muscle. In this model, thin filaments are mechanically severed from their connections to the Z-line and may then slide freely over the myosin filament when activated. The model is similar to the in vitro motility assay in that untethered actin filaments slide over myosin, but here the natural filament lattice is retained: sliding takes place through the lattice of thick filaments. We find, in this model, that while the extent of thin filament translation is variable from sarcomere to sarcomere, filaments never translate far enough to enter the opposite I-band. Unlike the in vitro motility assay, where the actin filament translates over the entire thick filament even with “incorrectly” polarized crossbridges as the sole driver, in this intact filament-lattice model, cross-bridges are apparently unable to move filaments in both directions. We also find that the pattern of filament translation is collective. Although the extent of translation may vary among sarcomeres, in any given half-sarcomere all actin filaments translate by the same degree. Further, the extent of translation is is the same in both halves of a given sarcomere. In rare instances where the extent of translation exhibited a transverse gradient across the myofibrillar half-sarcomere, the gradient was similar on both sides of the sarcomere. Filament translation within the sarcomere is thus collective. Some mechanism ensures that nearby but distinctly separated actin filaments move together and that cooperative-like behavior therefore extends to the supramolecular level.
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  • 198
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    Cell Motility and the Cytoskeleton 32 (1995), S. 187-204 
    ISSN: 0886-1544
    Keywords: neuronal growth cone migration ; filopodial dynamics ; simulation model ; computer graphics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The neuronal growth cone plays a fundamental role in nerve development and regeneration. A sensory-motile structure, it determines the path of axonal extension through its interactions with the extracellular environment, ultimately directing the formation of functional connections in the nervous system. Though several mechanisms of interaction have been proposed, these have been difficult to describe quantitatively due to the complexity of growth cone behavior, as manifested in the randomly and rapidly changing shape of the growth cone. The application of mathematical techniques to model growth cone shape and motility in terms of underlying processes represents a promising approach with untapped potential for helping to unravel this complexity while revealing new insights into axonal pathfinding events. This paper presents a simulation model for filopodial dynamics, a primary feature of the motile growth cone. The model produces realizations of dynamic filopodial structure on representative growth cones for a given set of model parameters, which include the rates of filopodial initiation, extension, and retraction, filopodial length at maximum extension, and angular orientation. These parameters are based on recent experimental characterization of filopodial dynamics [Buettner et al., 1994: Dev. Biol. 163:407-422]. The mathematical relationship between the model parameters and average filopodial number and length per growth cone is described, and the contribution of individual parameters to overall filopodial morphology is illustrated both visually and numerically. In addition, the model is used to simulate filopodial encounter with a target for various conditions of filopodial dynamics. The result is characterized in terms of a mean encounter time for a population of growth cones and provides an indication of the effect of individual parameters of filopodial dynamics on the encounter process. Future experimental testing will be required to develop the model further. However, in its current form, the model enables a first approximation analysis of many hypotheses of growth cone migration and pathfinding and offers insight into the the underlying mechanismes of nerve growth and regeneration. © 1995 Wiley-Liss, Inc.
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  • 199
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    Cell Motility and the Cytoskeleton 32 (1995), S. 245-257 
    ISSN: 0886-1544
    Keywords: actin ; cytochalasin ; microfilaments ; microtubules ; mitosis ; mitotic apparatus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK1 cells were treated with 10 μg/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana-like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 μg/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed that most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revelaed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosome congression with most chromosomes aligning at the metaphase plate after 10-15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti-tubulin staining of a monolayer of cells treated with 10 μg/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 258-272 
    ISSN: 0886-1544
    Keywords: adhering junctions ; desmosome ; assembly ; phosphorylation ; protein interaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have analyzed the kinetics of synthesis, phosphorylation, and stability of the soluble and insoluble plakoglobin (PG) and their interactions with Dsg1 and E-cadherin in Madin-Darby canine kidney (MDCK) epithelial cells in the absence of cell adhesion and after the induction of cell-cell contact. Using a combination of biochemical and morphological approaches, we show that newly synthesized PG enters a soluble:insoluble pool of proteins in a 60:40 ratio regardles of cell-cell contact. Following synthesis, PG is increasingly found in the insoluble pool. Although cell-cell contact does not effect either the size of each pool or the rate or efficiency of the transfer from the soluble into the insoluble pool, it results in a significant increase in the metabolic stability of the newly synthesized insoluble PG. The soluble PG initially forms separate complexes with E-cadherin and Dsg1. PG-Dsg1 complexes become insoluble and localize to the desmosome. PG-E-cadherin complexes remain soluble and are distributed intracellularly. The insoluble PG and E-cadherin detected at the cell periphery remain distinctly separate, as demonstrated previously [Hinck et al., 1994: J. Cell Biol. 125:1327-1340; Nathke et al., 1994: J. Cell Biol. 125:1341-1352]. In addition, we detected a separate pool of PG which is not associated with either Dsg1 or E-cadherin and after the induction of cell-cell contact becomes primarily insoluble and is distributed along the lateral membrane. Phoshorylation analysis showed that there is a significantly greater amount of phosphorylated PG in the soluble pool than in the insoluble pool. In addition the soluble pool is both serine and theronine phosphorylated, whereas the insoluble PG is primarily phosphorylated on serine residues. © 1995 Wiley-Liss, Inc.
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