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  • 1990-1994  (15,349)
  • 1955-1959  (1,513)
  • Cell & Developmental Biology  (9,440)
  • Biochemistry and Biotechnology  (4,158)
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  • transformation
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Year
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  • 1
    Electronic Resource
    Electronic Resource
    Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants (1994)
    Springer
    Plant cell reports 13 (1994), S. 130-134 
    Details
    ISSN: 1432-203X
    Keywords: Sinapis alba L. ; Agrobacterium tumefaciens ; transformation ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and β-glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical β glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00239878
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  • 2
    Electronic Resource
    Electronic Resource
    Isolation and transformation of rice aleurone protoplasts (1994)
    Springer
    Plant cell reports 13 (1994), S. 394-396 
    Details
    ISSN: 1432-203X
    Keywords: Rice ; α-amylase ; protoplasts ; aleurone ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234145
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  • 3
    Electronic Resource
    Electronic Resource
    Hairy roots of Brassica napus: II. Glutamine synthetase overexpression alters ammonia assimilation and the response to phosphinothricin (1994)
    Springer
    Plant cell reports 14 (1994), S. 41-46 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium rhizogenes ; Brassica napus ; glutamine synthetase ; phosphinothricin ; rape ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Hairy roots of Brassica napus (rape cv. Giant) have been produced that contain the cytosolic glutamine synthetase (GS) gene from Glycine max (soybean). Leaf explants were cocultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pLN16. This vector was constructed by inserting a soybean cytosolic GS cDNA into the multiple cloning site of pGA643, placing it under the control of the CaMV promoter. In addition, the T-DNA region of pLN16 contained a NPTII gene for selection of transformed cells. Transgenic hairy roots grew prolifically on hormone-free media containing a selective level of kanamycin. Southern and northern analyses confirmed the presence of soybean GS DNA and transcripts, respectively. These transformed hairy roots also have a greater abundance of the GS polypeptide, approximately 3–6 fold greater GS activity and lower levels of endogenous ammonia. Hairy roots provide a useful system for studying responses to phosphinothricin (PPT). Hairy roots grown in media containing PPT had lower GS activity, greater ammonia accumulation and slower growth than controls. The presence of the soybean GS gene in the hairy roots reduced these PPT-induced effects and resulted in higher GS activity, lower ammonia levels and faster growth than in PPT-treated controls. Greater tolerance of PPT was also seen in shoots regenerated from the hairy roots displaying elevated levels of GS activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233296
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  • 4
    Electronic Resource
    Electronic Resource
    Genetic ecology: A new interdisciplinary science, fundamental for evolution, biodiversity and biosafety evaluations (1994)
    Springer
    Cellular and molecular life sciences 50 (1994), S. 429-437 
    Details
    ISSN: 1420-9071
    Keywords: Genetics ; ecology ; DNA-transfer ; conjugation ; transformation ; transduction ; transposons ; dormant cells ; epilithon ; microbial colonisation ; symbiosis ; virus resistance ; biosafety ; release of genes ; insults to humanity ; evolution ; biodiversity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Genetic ecology is the extension of our modern knowledge in molecular genetics to studies of viability, gene expression and gene movements in natural environments like soils, aquifers and digestive tracts. In such milieux, the horizontal transfer of plasmid-borne genes between phylogenetically distant species has already been found to be much more frequent than had been expected from laboratory experience. For the study of exchanges involving chromosomally-located genes, more has to be learned about the behaviour of transposons in such environments. The results expected from studies in genetic ecology are relevant for considerations of evolution, biodiversity and biosafety. The role of this new field of research in restoring popular confidence in science and in its biotechnological applications is stressed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01920741
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  • 5
    Electronic Resource
    Electronic Resource
    Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA (1994)
    Springer
    Plant cell reports 14 (1994), S. 59-64 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; transformation ; T-DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233300
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  • 6
    Electronic Resource
    Electronic Resource
    Plasmid mediated silver resistance in Acinetobacter baumannii (1994)
    Springer
    BioMetals 7 (1994), S. 49-56 
    Details
    ISSN: 1572-8773
    Keywords: Acinetobacter ; conjugation ; curing ; plasmid ; silver uptake ; silver resistance ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10−6 recepient−1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5α cells at a frequency of 1 × 10−8 recepient−1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5α. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5α (pUPI199) effluxed 63% of the accumulated silver ions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00205194
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  • 7
    Electronic Resource
    Electronic Resource
    Transmission of injected DNA sequences to multiple eggs of Metaseiulus occidentalis and Amblyseius finlandicus (Acari: Phytoseiidae) following maternal microinjection (1994)
    Springer
    Experimental and applied acarology 18 (1994), S. 319-330 
    Details
    ISSN: 1572-9702
    Keywords: Maternal microinjection ; transformation ; genetic improvement
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The persistence of DNA injected into two species of adult female phytoseiids and its transmission to serial eggs deposited by them was assessed by the polymerase chain reaction (PCR). The effect of DNA concentration on persistence and transmission was examined in Metaseiulus occidentalis. M. occidentalis females were microinjected with plasmid DNA at three different concentrations (250, 500, 750 ng μL−1) and allowed to deposit one to five eggs before the females and their last eggs were analyzed. Plasmid DNA was found in 82% of the females assayed and in 70% of all the eggs analyzed (including the fifth eggs produced after microinjection). Transmission of DNA to multiple eggs was also examined in Amblyseius finlandicus. Females of this species are less traumatized by microinjection allowing analysis of transmission over a more extended number of eggs. Females were microinjected and allowed to deposit eggs until their death. DNA from every fifth egg was analyzed by the PCR. PCR products were amplified from 51% of the eggs and from all egg classes except the 30th egg. The persistence and presence of plasmid DNA in both eggs and females suggests that (1) maternal microinjection is a more efficient method for DNA delivery than traditional egg microinjection, (2) it may be possible to isolate transformants from fewer maternally-microinjected females than originally expected, and (3) maternal microinjection could be useful as a DNA delivery system in other phytoseiids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00116313
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  • 8
    Electronic Resource
    Electronic Resource
    Canonical correlation and reduction of multiple time series (1994)
    Springer
    Annals of the Institute of Statistical Mathematics 46 (1994), S. 625-631 
    Details
    ISSN: 1572-9052
    Keywords: Rank of multiple time series ; transformation ; spectrum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mathematics
    Notes: Abstract It is shown that a degenerate rankd-variate stationary time series can be reduced to a full rank time series of lower dimension via an orthogonal transformationT provided that ρ, the canonical correlation between past and future of the time series is strictly less than one. Procedures for estimation of rank of the multiple time series,T and testing ρ=1 are outlined, the latter is related to testing the unit root hypothesis in ARMA models.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00773471
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  • 9
    Electronic Resource
    Electronic Resource
    Genetics and gibberellin production inGibberella fujikuroi (1994)
    Springer
    Antonie van Leeuwenhoek 65 (1994), S. 217-225 
    Details
    ISSN: 1572-9699
    Keywords: Gibberella fujikuroi ; gibberellins ; mutants ; regulation by nitrogen ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development. The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00871950
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  • 10
    Electronic Resource
    Electronic Resource
    Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.) (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 259-264 
    Details
    ISSN: 1573-5044
    Keywords: monocotyledons ; Oryza sativa L. ; plant regeneration ; rice ; somatic embryogenesis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037729
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  • 11
    Electronic Resource
    Electronic Resource
    Factors that effect leaf regeneration efficiency in apple, and effect of antibiotics in morphogenesis (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 257-269 
    Details
    ISSN: 1573-5044
    Keywords: adventitious shoots ; Malus x domestica Borkh. ; tissue culture ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Several factors that affect the frequency of organogenesis in apple leaf explants were examined for the scion cultivars ‘Empire’, ‘Freedom’, ‘Golden Delicious’, ‘Liberty’, ‘McIntosh’, and ‘Mutsu’ and for the rootstocks Malling 7A and Malling 26. The main factors affecting morphogenesis were BA concentration, basal medium, leaf explant origin and maturity, explant orientation, and photosynthetic photon flux. Depending on the genotype, optimal regeneration was obtained using either 22.2 or 31.1 μM BA and the N6 basal medium, with the exception of ‘Golden Delicious’ which regenerated better on MS medium. After 6 weeks, the average number of shoots per segment varied from 5 to 16, and the percentage of regeneration between 70 and 100%, depending on the genotype tested and the maturity of the explant. Regeneration capacity increased dramatically from the tip towards the base of the leaf, and was higher from the middle to the proximal end. Cefotaxime and carbenicillin, two antibiotics commonly used during transformation studies to eliminate Agrobacterium tumefaciens from plant tissue, were tested to determine their effect on morphogenesis. Cefotaxime at a dose of 250 mg 1-1 enhanced regeneration and shoot development, whereas carbenicillin at a dose of 500 mg l-1 induced abundant callus formation and inhibited regeneration. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. Schemes for selection and recovery of transgenic apple plants when kanamycin is used as the selection agent are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00042339
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  • 12
    Electronic Resource
    Electronic Resource
    A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain (1994)
    Springer
    Photosynthesis research 42 (1994), S. 121-131 
    Details
    ISSN: 1573-5079
    Keywords: chloroplast ; Photosystem II ; psbA ; site-directed mutagenesis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5′- and 3′-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe r) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe r phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (QA) to secondary quinone (QB) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (QA −).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02187123
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  • 13
    Electronic Resource
    Electronic Resource
    Micropropagation of thirteen Malus cultivars and rootstocks, and effect of antibiotics on proliferation (1994)
    Springer
    Plant growth regulation 15 (1994), S. 55-67 
    Details
    ISSN: 1573-5087
    Keywords: apple (Malus × domestica Borkh.) ; selection ; tissue culture ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Several factors that affect in vitro establishment, proliferation, and rooting of thirteen Malus cultivars and rootstocks were studied. Apple shoot tips (1.5±0.5 cm in length) were established using ascorbic and citric acids as antioxidants. Four proliferation media containing 1.0 mg 1−1 BA and different concentrations of IBA and GA3 were tested. Proliferation rates varied depending on the genotype and medium used. The highest proliferation rate was obtained for a rootstock that produced 11.6±2.5 shoots (1.5±0.8 cm in length) per tube per month. Rooting was induced with IBA for all the genotypes tested. The optimal IBA concentration was cultivar dependent (between 0.1 and 1.0 mg 1−1 IBA), and lower concentrations were necessary to induce rooting in liquid rather than in solid medium. The effects on shoot-tip proliferation of cefotaxime, carbenicillin and kanamycin, three antibiotics commonly used for transformation studies, were also evaluated. Cefotaxime at 200 mg 1−1 stimulated shoot growth and development, but at 500 mg 1−1 caused abnormal shoot morphology. Carbenicillin at 500 mg 1−1, alone or in combination with cefotaxime at 200 mg 1−1, inhibited proliferation and caused excessive enlargement of the basal leaves, inducing callus formation and release of phenolic compounds in the medium. Kanamycin at 50 mg 1−1 was phytotoxic and caused shoot chlorosis and necrosis. Consideration of the toxicity of these antibiotics is critical when designing transformation schemes for selection and recovery of transgenic apple plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00024677
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  • 14
    Electronic Resource
    Electronic Resource
    Production of fertile transgenic maize by electroporation of suspension culture cells (1994)
    Springer
    Plant molecular biology 24 (1994), S. 51-61 
    Details
    ISSN: 1573-5028
    Keywords: Zea mays L. ; transformation ; electroporation ; bar ; phosphinothricin acetyltransferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Fertile, transgenic maize plants were generated by electroporation of suspension culture cells that were treated with a pectin-degrading enzyme. Electroporation of cells from two different suspension cultures, one derived from A188 X B73 and one derived from a B73-related inbred, with a plasmid containing the bar gene, resulted in high-frequency recovery of stably transformed callus lines. Plants were regenerated from thirteen transformed callus lines and transmission of bar to progeny was demonstrated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040573
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  • 15
    Electronic Resource
    Electronic Resource
    Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.) (1994)
    Springer
    Plant molecular biology 24 (1994), S. 401-405 
    Details
    ISSN: 1573-5028
    Keywords: Lolium perenne L. ; transformation ; rice gene GOS2 ; long-term GUS expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×106 cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020178
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  • 16
    Electronic Resource
    Electronic Resource
    Molecular improvement of cereals (1994)
    Springer
    Plant molecular biology 25 (1994), S. 925-937 
    Details
    ISSN: 1573-5028
    Keywords: cereals ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014667
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  • 17
    Electronic Resource
    Electronic Resource
    Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment (1994)
    Springer
    Plant molecular biology 25 (1994), S. 951-961 
    Details
    ISSN: 1573-5028
    Keywords: Cell biology ; epigenetics ; maize ; transformation ; transgenes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014669
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  • 18
    Electronic Resource
    Electronic Resource
    Stably transformed cell lines from protoplasts of maize endosperm suspension cultures (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 39-46 
    Details
    ISSN: 1573-5044
    Keywords: endosperm cell culture ; maize ; protoplast ; transformation ; zeins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were isolated from Zea mays (L.) A69Y endosperm suspension cultures and transformed by polyethylene glycol mediated DNA uptake with chimaeric gene constructs containing β-glucuronidase (GUS) or neomycin phosphotransferase II (NPTII); GUS-expressing and Kanamycin-resistant cultures were recovered. The transformed cells showed integration of the introduced foreign genes into genomic DNA and maintained their ability to synthesize endosperm-specific reserve proteins (zeins). No deletion or rearrangement of zein genes were observed in transformed cultures. Stable transformation of cultured maize endosperm cells may therefore represent a new methodological approach for the study of the transcriptional regulation of endosperm-expressed genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048115
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  • 19
    Electronic Resource
    Electronic Resource
    A simple technique for producing 13C or 14C-labelled fronds of Lemna gibba and their use in soil incubation investigations (1994)
    Springer
    Plant and soil 160 (1994), S. 185-191 
    Details
    ISSN: 1573-5036
    Keywords: carbon-labelling ; carbon dioxide production ; decomposition ; 14C-glucose ; Lemna ; soil organic matter ; sugars ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The duckweed Lemna gibba required light and a suitable energy source such as sucrose, glucose or fructose, for maximum growth in culture. The requirement for light was relatively unimportant and the plants grew well in a photon flux density of only 52 μmol m-2s-1 PAR. The uptake and incorporation of uniformly labelled 14C-glucose into fronds was related only to the concentration of the sugar. When incubated with soil, labelled L. gibba behaved in a manner similar to that of labelled ryegrass roots which had been produced by a more elaborate technique using a 14CO2 labelled atmosphere. During incubation with soil for 224 days the L. gibba material (specific activity 6133 Bq mg-1 d. wt) lost 64% of its radioactivity as 14CO2 and ryegrass (specific activity 6634 Bq mg-1 d. wt) lost 49%. Alkaline extracted humic and fulvic acids from soil had specific activities for the L. gibba incubation of 3409 and 407 Bq mg-1 solid and for ryegrass roots of 4609 and 546 Bq mg-1 solid respectively. The production of 13C or 14C-labelled L. gibba can be undertaken using only simple equipment producing material the specific radioactivity of which can be controlled by adjusting the activity of the sugar energy source.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00010144
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  • 20
    Electronic Resource
    Electronic Resource
    Cloning of ω3 desaturase from cyanobacteria and its use in altering the degree of membrane-lipid unsaturation (1994)
    Springer
    Plant molecular biology 26 (1994), S. 249-263 
    Details
    ISSN: 1573-5028
    Keywords: desB gene ; desaturase ; fatty acid ; Synechococcus sp. PCC 7942 ; Synechocystis sp. PCC 6803 ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cyanobacteria respond to a decrease in temperature by desaturating fatty acids of membrane lipids to compensate for the decrease in membrane fluidity. Among various desaturation reactions in cyanobacteria, the desaturation of the ω3 position of fatty acids is the most sensitive to the change in temperature. In the present study, we isolated a gene, designated desB, for the ω3 desaturase from the cyanobacterium, Synechocystis sp. PCC 6803. The desB gene encodes a protein a 359 amino-acid residues with molecular mass of 41.9 kDa. The desB gene is transcribed as a monocistronic operon that produced a single transcript of 1.4 kb. The level of the desB transcript in cells grown at 22°C was 10 times higher than that in cells grown at 34°C. In order to manipulate the fatty-acid unsaturation of membrane lipids, the desB gene in Synechocystis sp. PCC 6803 was mutated by insertion of a kanamycin-resistance gene cartridge. The resultant mutant was unable to desaturate fatty acids at the ω3 position. The desA gene, which encodes the Δ12 desaturase of Synechocystis sp. PCC 6803, and the desB gene were introduced into Synechococcus sp. PCC 7942. Whilst the parent cyanobacterium can only desaturate membrane lipids at the Δ9 position of fatty acids, the resultant transformant was able to desaturate fatty acids of membrane lipids at the Δ9, Δ12 and ω3 positions. These results confirm the function of the desB gene and demonstrate that it is possible to genetically manipulate the fatty-acid unsaturation of membrane lipids in cyanobacteria.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00039536
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  • 21
    Electronic Resource
    Electronic Resource
    Identification of conserved domains in the Δ12 desaturases of cyanobacteria (1994)
    Springer
    Plant molecular biology 24 (1994), S. 643-650 
    Details
    ISSN: 1573-5028
    Keywords: Anabaena variabilis ; fatty-acid desaturation ; Synechococcus PCC7002 ; Synechococcus PCC7942 ; Synechocystis PCC6714 ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cyanobacterial genes for enzymes that desaturate fatty acids at the Δ12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the Δ12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of ω3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023560
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  • 22
    Electronic Resource
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    Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex (1994)
    Springer
    Plant molecular biology 24 (1994), S. 779-788 
    Details
    ISSN: 1573-5028
    Keywords: Chlamydomonas reinhardtii ; chloroplast ; transformation ; photosystem II ; psbK
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029859
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  • 23
    Electronic Resource
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    Cotton (Gossypium hirsutum L.) pollen-specific polygalacturonase mRNA: tissue and temporal specificity of its promoter in transgenic tobacco (1994)
    Springer
    Plant molecular biology 26 (1994), S. 1989-1993 
    Details
    ISSN: 1573-5028
    Keywords: polygalacturonase ; pollen-specific promoter ; cotton ; transgenics ; transformation ; β-glucuronidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A gene (G9) expressed during late microsporogenesis in cotton (Gossypium hirsutum L.) was isolated. Sequence analysis of the cDNA (1.3 kb) as well as the gene (2.6 kb) revealed an open reading frame of 1233 bases encoding a protein of 43.9 kDa. The coding region of the gene is interrupted by three introns. Northern analysis of the RNA from developing anthers showed that the transcripts appear 12 days before anthesis and that the maximal concentration of RNA occurs in pollen on the day of anthesis. This pattern of gene expression suggests functions in post-anthesis events. Sequence comparisons with other known plant genes indicated that G9 is homologous to polygalacturonases. The G9 promoter conferred tissue and temporal specificity of β-glucuronidase (GUS) expression in transgenic tobacco plants. Thus, the G9 promoter can be used to drive gene expression in homologous as well as heterologous plants in a tissue-specific manner.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019509
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  • 24
    Electronic Resource
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    Cellular microtubules heterogeneous in their content of microtubule-associated protein 4 (MAP4) (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 133-149 
    Details
    ISSN: 0886-1544
    Keywords: MAP4 depletion ; antibody blocking ; detyrosination ; midbody ; asymmetric cell processes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of Mr ∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270205
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  • 25
    Electronic Resource
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    Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 193-205 
    Details
    ISSN: 0886-1544
    Keywords: amoeboid motility ; fluorescence ratio imaging ; BCECF ; nematodes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270302
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  • 26
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    Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 248-261 
    Details
    ISSN: 0886-1544
    Keywords: myogenesis ; protein isoforms ; muscle types ; Z-disc ; phosphorylation ; chicken muscle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin-binding protein (ABP) filamin is associated with stress fiber-like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z-disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z-disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two-dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non-striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14-somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270306
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    Regulation and evolution of the single alpha-tubulin gene of the ciliate Tetrahymena thermophila (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 272-283 
    Details
    ISSN: 0886-1544
    Keywords: cell cycle ; transcription ; mRNA decay ; autoregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270308
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    Microtubule centers and the interphase microtubule cytoskeleton in amoebae of the cellular slime molds (mycetozoans) Acytostelium leptosomum and Protostelium mycophaga (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 45-58 
    Details
    ISSN: 0886-1544
    Keywords: MTOC ; cytoplasmic microtubule complex ; antitubulin ; Immunofluorescence ; ultrastructure ; immunogold ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron-opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus-associated and “remote” MTCs are structurally identical. They consist of a ring-shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280105
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  • 29
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    Movement of Myzostomum spermatozoa: Calcium ion regulation of swimming direction (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 135-142 
    Details
    ISSN: 0886-1544
    Keywords: bidirectional swimming ; flagellar movement ; helical bends ; 9+0 axoneme ; planar bends ; viscosity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spermatozoa of the small myzostomid worm Myzostomum cirriferum usually swim with the flagellum foremost but occasionally stop and then swim with the head foremost. The spermatozoa have axoneme of the 9+0 type; thus each lacks the central pair microtubules. The flagellum emerges in the anterior end of the cell body and attaches to it with junctions. To understand the mechanism regulating the swimming direction of the spermatozoa, we recorded the sperm and their flagellar movements using a video camera with a high-speed shutter. The effects of calcium and viscosity on these movements were also examined.The cell body with the flagellum attached to it formed a curved plate during beating, while the free portion of the flagellum beats with small helical bends. Motive force to propel a spermatozoon was mainly due to the bends in the cell body. The spermatozoa reversed the direction of their swimming as a result of a change in the direction of bend propagation. The direction of bend propagation was regulated by calcium; the bends in the cell body propagated from the end of the head toward the free portion of the flagellum at low concentrations of Ca2+, whereas the direction of bend propagation was reversed at high concentrations of this ion. High viscosity of the medium stimulated a change in the direction of bend propagation. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280205
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  • 30
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    Role of tropomyosin, α-actinin, and actin binding protein 280 in stabilizing triton insoluble f-actin in basal and chemotactic factor activated neutrophils (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 155-164 
    Details
    ISSN: 0886-1544
    Keywords: microfilamentous cytoskeleton ; actin binding proteins ; formyl peptides ; ionic extraction ; immunoblots ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: F-actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F-actin exists in two structurally and functionally distinct pools: Triton insoluble F-actin (TIF)-cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F-actin (TSF)-unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F-actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F-actin pool, the distribution and quantity of three candidate regulatory proteins, α-actinin, tropomyosin (TM), and actin binding protein (ABP-280), were compared in F-actin (Triton insoluble and Triton soluble) and G-actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F-actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) α-actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α-actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F-actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F-actin pool in temporal association with F-actin polymerization in the Triton insoluble F-actin pool. Shifts in ABP 280 or α-actinin do not occur. The results suggest that TM, α-actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280207
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    Characterization of keratin densities in mitotic wish cells (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 165-178 
    Details
    ISSN: 0886-1544
    Keywords: WISH ; Keratin ; 3-D reconstruction ; mitosis ; intermediate filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Three dimensional (3-D) reconstruction of four mitotic WISH cells from ultrathin sections gave an informative representation of the spatial distribution of keratin densities in these cells. The correspondence between the densities as studied by transmission electron microscopy (TEM) and the Keratin bodies initially revealed by immunoflourescent colabeling of cultures, was confirmed by immunoelectron-microscopy. The smaller, and sometimes more elongated densities, were relatively abundant just beneath the subplasmalemmal microfilament band; and at certain levels of the mitotic cell they were observed to be connected to neighboring densities by intact intermediate filaments (IFs). The larger and more spherical densities appeared to be somewhat more discrete and randomly distributed. Other observed associations of the keratin densities included the telophase contractile ring of microfilaments, chromosomes, the reformed telophase nucleus, and desmosomal junctions with neighboring interphase cells. Cytochalasin D (CD) treatment of cells displaced the peripheral keratin densities toward the cell membrane. The density volume constituted 0.52% to 1.57% of the total cell volume, and the proportional density size was decreased in the cells that had progressed into anaphase and telophase. The observed formation and subsequent dissolution of keratin densities during mitosis may represent a dynamic mechanism of restructuring the keratin cytoskeleton in an unpolymerized form in order to allow for rapid reformation of interphase cell junctions. The physical associations observed between intact IFs and the keratin densities may provide support at certain depths of the mitotic cell, and the juxtaposition of densities with nuclear components suggests a possible source of and role for keratin IFs during nuclear events. © 1994 Wiley-Liss, Inc.
    Additional Material: 12 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280208
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    Control of flagellar bending: A new agenda based on dynein diversity (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 199-204 
    Details
    ISSN: 0886-1544
    Keywords: axoneme ; cilia ; flagella ; microtubule ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Observations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley-Liss, Inc.
    Additional Material: 1 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280303
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  • 33
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    Cloning and sequencing of cDNAs encoding the actin cross-liking protein transgelin defines a new family of actin-associated proteins (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 243-255 
    Details
    ISSN: 0886-1544
    Keywords: cytoskeleton ; actin binding ; transgelin sequence ; gelation ; gene family ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065-1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT-PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987: J. Biol. Chem. 262:5985-5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518-530], and human WS3-10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1-7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal-Ginard, 1991: J. Biol. Chem. 266:13284-13288], and Drosophila mp20 [Ayme-Southgate et al., 1989: J. Cell Biol. 108:521-531] suggest that all of these proteins may be classified as members of a new transgelin multigene family. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280307
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  • 34
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    Signal transduction and calcium: A suggested role for the cytoskeleton in inositol 1,4,5-trisphosphate action (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 279-284 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280402
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  • 35
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    Coordinated expression of five tropomyosin isoforms and β-actin in astrocytes treated with dibutyryl cAMP and cytochalasin D (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 303-316 
    Details
    ISSN: 0886-1544
    Keywords: cytoskeleton ; cell culture ; gene expression ; Northern blot ; serum-induction ; rat ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytochalasin D and dBcAMP cause cultured astrocytes to change from flat cells to retrated process-bearing cells. F-actin was present throughout cells stimulated with dBcAMP for 16 h, whereas cytochalasin D caused F-actin to form massive aggregates at the tips of the cell processes. The two drugs differently regulated the expression of both β-actin and tropomyosin genes in astrocytes cultured in the presence or absence of serum: dBcAMP caused down-regulation and cytochalasin D caused up-regulation. Northern blot analyses indicated that: (1) serum deprivation halved the concentration of all tropomyosin transcripts (TM-1, TM-2, TM-4, TMBr-1, TMBr-2). Serum induced TM-4 via transcriptional activation, independent of protein synthesis, (2) dBcAMP induced down-regulation of β-actin (-50%) and tropomyosin transcripts (-35 to 52%) even in the presence of serum. The concentration of profilin mRNA decreased in dBcAMP-reactive astrocytes (-46%). The decrease in β-actin mRNA concentration was not blocked by cycloheximide, whereas down-regulation of tropomyosin transcripts was completely reversed when protein synthesis was inhibited, and (3) cytochalasin D induced an increase in the concentration of tropomyosin transcripts (+ 69 to 185%) which was cumulative with serum stimulation. Cytochalasin D induction of both β-actin and TM-4 operated through transcriptional activation, independent of protein synthesis.The production of all tropomyosin transcripts examined here were strictly coordinated with β-actin expression in serum-, dBcAMP- and cytochalasin D-treated astrocytes. This indicates that the differential expression of tropomyosin isoforms occurring during astrocyte maturation is due to more complex regulation than that involved in serum- or cAMP-stimulated astrocytes. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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    Metachronal activity of cultured mucociliary epithelium under normal and stimulated conditions (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 333-345 
    Details
    ISSN: 0886-1544
    Keywords: ciliary beat frequency ; metachronal wave ; ciliary coupling ; extracellular ATP ; acetylcholine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated.On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5-9 μm, and the direction of the wave propagation was in the range of 90°-125° clockwise to the ESD.When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10°-30°, and the metachronal wavelength remained unaltered. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280407
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    Assembly and bundling of marginal band microtubule protein: Role of tau (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 57-71 
    Details
    ISSN: 0886-1544
    Keywords: microtubule bundling ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 μm in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. © 1994 Wiley-Liss, Inc.
    Additional Material: 15 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290106
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    Striated fibers in trichomonads: Costa proteins represent a new class of proteins forming striated roots (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 82-93 
    Details
    ISSN: 0886-1544
    Keywords: trichomonads ; cytoskeleton ; antibodies ; electrophoresis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The production of monoclonal antibodies and the use of biochemical techniques revealed that B-type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A-type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein of Trichomonas vaginalis also recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A- or B-type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root-forming proteins in addition to centrin, giardin, and assemblin. © 1994 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290108
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    Microtubule associated protein MAP1A is an actin-binding and crosslinking protein (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 110-116 
    Details
    ISSN: 0886-1544
    Keywords: high-molecular weight MAPs ; microfilaments ; microtubules ; low-shear viscometry ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: High molecular weight microtubule-associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G- and F-actin. Using a solid-phase immunoassay we show that MAP1A binds in a dose-dependent manner to both G-actin and F-actin. Addition of MAP1A to F-actin causes gelation of F-actin and SDS-PAGE analysis shows that MAP1A co-sediments with the gelled network, under conditions where F-actin alone does not pellet. The low apparent viscosity of F-actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F-actin. Co-incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non-neuronal cellular morphology. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290203
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    A model of flagellar and ciliary functioning which uses the forces transverse to the axoneme as the regulator of dynein activation (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 141-154 
    Details
    ISSN: 0886-1544
    Keywords: dynein arms ; nexin links ; radial spokes ; relaxation oscillator ; doublet microtubules ; biological oscillators ; computer model ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ciliary and flagellar motion is driven by the dynein-tubulin interaction between adjacent doublets of the axoneme, and the resulting sliding displacements are converted into axonemal bends that are propagated. When the axoneme is bent in the normal beating plane, force develops across the axoneme in the plane of the bend. This transverse force (t-force) has maximal effect on the interdoublet spacing of outer doublets 2-4 on one side of the axoneme and doublets 7-9 on the opposite side. Episodes of sliding originates as the t-force brings these doublets into closer proximity (allowing dynein bridges to form) and are terminated when these doublets are separated from each other by the t-force. A second factor, the adhesive force of the dynein-tubulin attachments (bridges), also acts to pull neighboring doublets closer together. This force resists termination of a sliding episode once initiated, and acts locally to give the population of dynein bridges a type of excitability. In other words, as bridges form, the probability of nearby bridges attaching is increased by a positive feedback exerted through the interdoublet spacing. A conceptual working hypothesis explaining the behavior of cilia and flagella is proposed based on the above concepts. Additionally, the feasibility of this proposed mechanism is demonstrated using a computer simulation. The simulation uses a Monte Carlo-type algorithm for dynein attachment and adhesive force, together with a geometric evaluation of the t-force on the key microtubule pairs. This model successfully develops spontaneous oscillations from any starting configuration (including a straight position). It is compatible with the physical dimensions, mechanical properties and bridge forces measured in real cilia and flagella. In operation, it exhibits many of the observed actions of cilia and flagella, most notably wave propagation and the ability to produce both cilia-like and flagella-like waveforms. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290206
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    Modulation of fibroblastic cytoskeletal features during pathological situations: The role of extracellular matrix and cytokines (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 195-203 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 3 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970290302
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    Characterization of a monoclonal antibody prepared against plant actin (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 339-344 
    Details
    ISSN: 0886-1544
    Keywords: microfilament ; phalloidin ; immunoblotting ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Anti-actin monoclonal antibodies were prepared using phalloidin-stabilized actin that was purified from pea roots by DNase I affinity chromatography. One monoclonal antibody, designated mAb3H11, bound plant actin in preliminary screenings and was further analyzed. Immunoblot analysis showed that this antibody had a high affinity for plant actin in crude and purified preparations but a low affinity for rabbit muscle actin. In immunoblots of plant extracts separated on two-dimensional gels it appeared to bind all actin isoforms recognized by the JLA20 anti-chicken actin antibody. Using immunofluorescent cytochemistry, the antibody was used to observe actin filaments in aldehyde-fixed and methanol-treated tobacco protoplasts. These results indicate that mAb3H11 should be a useful reagent for the study of plant actins. © 1994 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
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    Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 366-374 
    Details
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microfilaments ; cytochalasin ; gravity ; amiprophos-methyl ; tip-growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290409
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    Protein folding: Predicting predicting (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 1-3 
    Details
    ISSN: 0887-3585
    Keywords: protein folding ; protein conformation ; Paracelsus award ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 1 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340190102
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    Structure of trichosanthin at 1.88 Å resolution (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 4-13 
    Details
    ISSN: 0887-3585
    Keywords: trichosanthin ; ribosome-inactivating proteins ; crystal structure ; orthorhombic ; molecular replacement ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Trichosanthin (TCS) is one of the single chain ribosome-inactivating proteins (RIPs). The crystals of the orthorhombic form of trichosanthin have been obtained from a citrate buffer (pH 5.4) with KC1 as the precipitant. The crystal belongs to the space group P212121 with a = 38.31, b = 76.22, c = 79.21 Å. The structure was solved by molecular replacement method and refined using the programs XPLOR and PROLSQ to an R-factor of 0.191 for the reflections within the 6-1.88 Å resolution range. The bond length and bond angle in the protein molecule have root-mean-square deviations from ideal value of 0.013 Å and 3.3°, respectively. The refined model includes 247 residues and 197 water molecules. The TCS molecule consists of two structural domains. The large domain contains six α-helices, a six stranded sheet, and an antiparallel β-sheet. The small domain has a largest α-helix, which shows a distinct bend. The possible active site of the molecule located on the cleft between two domains was proposed. In the active site Arg-163 and Glu-160, Glu-189 and Arg-122 form two ion pairs, Glu-189 and Gln-156 are hydrogen bonded to each other. Three water molecules are bonded to the residues in the active site region. The structures of TCS molecule and ricin A-chain (RTA) superimpose quite well, showing that the structures of the two protein molecules are homologous. Comparison of the structures of the TCS molecule in this orthorhombic crystal with that in the monoclinic crystal indicates that there are no essential differences of the structures between the two protein crystals. © 1994 Wiley-Liss, Inc.
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    Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 80-83 
    Details
    ISSN: 0887-3585
    Keywords: maize protein ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P212121 with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.36 Å 3/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuKα and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.
    Additional Material: 1 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340190111
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  • 47
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    α-Helix-forming propensities in peptides and proteins (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 85-97 
    Details
    ISSN: 0887-3585
    Keywords: protein conformation ; secondary structure ; protein folding ; helix stability ; helix formation ; conformational entropy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Much effort has been invested in seeking to understand the thermodynamic basis of helix stability in both peptides and proteins. Recently, several groups have measured the helix-forming propensities of individual residues (Lyu, P. C., Liff, M. I., Marky, L. A., Kallenbach, N. R. Science 250:669-673, 1990; O'Neil, K. T., DeGrado, W. F. Science 250:646-651, 1990; Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M., Baldwin, R. L. Nature (London) 344:268-270, 1990). Using Monte Carlo computer simulations, we tested the hypothesis that these differences in measured helix-forming propensity are due primarily to loss of side chain conformational entropy upon helix formation (Creamer, T. P., Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 89:5937-5941, 1992). Our previous study employed a rigid helix backbone, which is here generalized to a completely flexible helix model in order to ensure that earlier results were not a methodological artifact. Using this flexible model, side chain rotamer distributions and entropy losses are calculated and shown to agree with those obtained earlier. We note that the side chain conformational entropy calculated for Trp in our previous study was in error; a corrected value is presented. Extending earlier work, calculated entropy losses are found to correlate strongly with recent helix propensity scales derived from substitutions made within protein helices (Horovitz, A., Matthews, J. M., Fersht, A. R. J. Mol. Biol. 227:560-568, 1992; Blaber, M., Zhang, X.-J., Matthews, B. M. Science 260:1637-1640, 1993). In contrast, little correlation is found between these helix propensity scales and the accessible surface area buried upon formation of a model polyalanyl α-helix. Taken in sum, our results indicate that loss of side chain entropy is a major determinant of the helix-forming tendency of residues in both peptide and protein helices. © 1994 Wiley-Liss, Inc.
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    Searching protein structure databases has come of age (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 165-173 
    Details
    ISSN: 0887-3585
    Keywords: algoriths ; structure alignment ; Protein Data Bank ; protein superfamilies ; structural homology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The number of protein structures known in atomic detail has increased from one in 1960 (Kendrew, J. C., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C., Shore, V. C. Nature (London) 185:422-427, 1960) to more than 1000 in 1994. The rate at which new structures are being published exceeds one a day as a result of recent advances in protein engineering, crystallography, and spectroscopy. More and more frequently, a newly determined structure is similar in fold to a known one, even when no sequence similarity is detectable. A new generation of computer algorithms has now been developed that allows routine comparison of a protein structure with the database of all known structures. Such structure database searches are already used daily and they are beginning to rival sequence database searches as a tool for discovering biologically interesting relationships. © 1994 Wiley-Liss, Inc.
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    HOOK: A program for finding novel molecular architectures that satisfy the chemical and steric requirements of a macromolecule binding site (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 199-221 
    Details
    ISSN: 0887-3585
    Keywords: multicopy simulation search ; rational drug design ; database search ; computer-aided design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A program (HOOK) is described for generating potential ligands that satisfy the chemical and steric requirements of the binding region of a macromolecule. Functional group sites with defined positions and orientations are derived from known ligand structures or the multicopy simulation search (MCSS) method (Miranker, A., Karplus, M. Proteins 11:29-34, 1991). HOOK places molecular “skeletons” from a database into the protein binding region by making bonds between sites (“hooks”) on the skeleton and functional groups. The nonpolar interactions with the binding region of candidate molecules are assessed by use of a simplified van der Waals potential. The method is illustrated by constructing ligands for the sialic acid binding site of the hemagglutinin from the influenza A virus and the active site of chloramphenicol acetyltransferase. Aspects of the HOOK program that lead to a highly efficient search of 105 or more skeletons for binding to 102 or more functional group minima are outlined. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340190305
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    Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 230-243 
    Details
    ISSN: 0887-3585
    Keywords: aspartate transcarbamylase ; multienzyme complex ; comparative structure modeling ; allosteric enzymes ; molecular evolution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley-Liss, Inc.
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340190401
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    Where is NMR taking us?This article is a US Government work and, as such, is in the public domain in the United States of America. (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 273-276 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340190402
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    Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 19 (1994), S. 302-309 
    Details
    ISSN: 0887-3585
    Keywords: dinuclear copper site ; hemocyanin ; oxygen binding ; allosteric regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The X-ray structure of an oxygenated hemocyanin molecule, subunit II of Limulus polyphemus hemocyanin, was determined at 2.4 Å resolution and refined to a crystallographic R-factor of 17.1%. The 73-kDa subunit crystallizes with the symmetry of the space group R32 with one subunit per asymmetric unit forming hexamers with 32 point group symmetry. Molecular oxygen is bound to a dinuclear copper center in the protein's second domain, symmetrically between and equidistant from the two copper atoms. The copper-copper distance in oxygenated Limulus hemocyanin is 3.6 ± 0.2 Å, which is surprisingly 1 Å less than that seen previously in deoxygenated Limulus polyphemus subunit II hemocyanin (Hazes et al., Protein Sci. 2:597, 1993). Away from the oxygen binding sites, the tertiary and quaternary structures of oxygenated and deoxygenated Limulus subunit II hemocyanins are quite similar. A major difference in tertiary structures is seen, however, when the Limulus structures are compared with deoxygenated Panulirus interruptus hemocyanin (Volbeda, A., Hol, W. G. J. J. Mol. Biol. 209:249, 1989) where the position of domain 1 is rotated by 8° with respect to domains 2 and 3. We postulate this rotation plays an important role in cooperativity and regulation of oxygen affinity in all arthropod hemocyanins. © 1994 Wiley-Liss, Inc.
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340200301
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    Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: Cytochrome P450eryF (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 197-201 
    Details
    ISSN: 0887-3585
    Keywords: cytochrome P450 ; erythromycin ; P450eryF ; crystallization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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    Pyrrolidone carboxyl peptidase (Pcp): An enzyme that removes pyroglutamic acid (pGlu) from pGlu-peptides and pGlu-proteins (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 34-51 
    Details
    ISSN: 0887-3585
    Keywords: enzymology ; protein structure ; biochemical properties ; gene characterization ; bacterial diagnosis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Pyrrolidone carboxyl peptidase (EC 3.4.11.8) is an exopeptidase commonly called PYRase, which hydrolytically removes the pGlu from pGlu-peptides or pGlu-proteins.pGlu also known as pyrrolidone carboxylic acid may occur naturally by an enzymatic procedure or may occur as an artifact in proteins or peptides. The enzymatic synthesis of pGlu suggests that this residue may have important biological and physiological functions. Several studies are consistent with this supposition.PYRase has been found in a variety of bacteria, and in plant, animal, and human tissues For over two decades, biochemical and enzymatic properties of PYRase have been investigated. At least two classes of PYRase have been characterized. The first one includes the bacterial and animal type I PYRases and the second one the animal type II and serum PYRases. Enzymes from these two classes present differences in their molecular weight and in their enzymatic properties.Recently, the genes of PYRases from four bacteria, have been cloned and characterized, allowing the study of the primary structure of these enzymes, and their over-expression in heterelogous organisms. Comparison of the primary structure of these enzymes revealed striking homologies.Type I PYRases and bacterial PYRases are generally soluble enzymes, whereas type II PYRases are membrane-bound enzymes. PYRase II appears to play as important a physiological role as other neuropeptide degrading enzymes. However, the role of type I and bacterial PYRases remains unclear.The primary application of PYRase has been its utilization for some protein or peptide sequencing. Development of chromogenic substrates for this enzyme has allowed its use in bacterial diagnosis. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340200106
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    Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 52-67 
    Details
    ISSN: 0887-3585
    Keywords: nuclear magnetic resonance ; defensin ; hydrogen exchange ; antimicrobial peptides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 102 to 105 times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340200107
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    A Connected-cluster of hydration around myoglobin: Correlation between molecular dynamics simulations and experiment (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 18 (1994), S. 133-147 
    Details
    ISSN: 0887-3585
    Keywords: myoglobin ; simulation ; hydration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An analysis of a molecular dynamics simulation of metmyoglobin in an explicit solvent environment of 3,128 water molecules has been performed. Both statics and dynamics of the protein-solvent interface are addressed in a comparison with experiment. Three-dimensional density distributions, temperature factors, and occupancy weights are computed for the solvent by using the trajectory coordinates. Analysis of the hydration leads to the localization of more than 500 hydration sites distributed into multiple layers of solvation located between 2.6 and 6.8 Å from the atomic protein surface. After locating the local solvent density maxima or hydration sites we conclude that water molecules of hydration positions and hydration sites are distinct concepts. Both global and detailed properties of the hydration cluster around myoglobin are compared with recent neutron and X-ray data on myoglobin. Questions arising from differences between X-ray and neutron data concerning the locations of the protein-bound water are investigated. Analysis of water site differences found from X-ray and neutron experiments compared with our simulation shows that the simulation gives a way to unify the hydration picture given by the two experiments. © 1994 John Wiley & Sons, Inc.
    Additional Material: 13 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340180206
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    Evaluation of the conformational free energies of loops in proteins (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 18 (1994), S. 119-132 
    Details
    ISSN: 0887-3585
    Keywords: electrostatics ; protein conformation ; DelPhi ; hydrophobicity ; RNase H ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In this paper we discuss the problem of including solvation free energies in evaluating the relative stabilities of loops in proteins. A conformational search based on a gas-phase potential function is used to generate a large number of trial conformations. As has been found previously, the energy minimization step in this process tends to pack charged and polar side chains against the protein surface, resulting in conformations which are unstable in the aqueous phase. Various solvation models can easily identify such structures. In order to provide a more severe test of solvation models, gas phase conformations were generated in which side chains were kept extended so as to maximize their interaction with the solvent. The free energies of these conformations were compared to that calculated for the crystal structure in three loops of the protein E. coli RNase H, with lengths of 7, 8, and 9 residues. Free energies were evaluated with a finite difference Poisson-Boltzmann (FDPB) calculation for electrostatics and a surface area-based term for nonpolar contributions. These were added to a gas-phase potential function. A free energy function based on atomic solvation parameters was also tested. Both functions were quite successful in selecting, based on a free energy criterion, conformations quite close to the crystal structure for two of the three loops. For one loop, which is involved in crystal contacts, conformations that are quite different from the crystal structure were also selected. A method to avoid precision problems associated with using the FDPB method to evaluate conformational free energies in proteins is described. © 1994 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340180205
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    Crystallization and structural analysis of bullfrog red cell L-subunit ferritins (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 18 (1994), S. 107-118 
    Details
    ISSN: 0887-3585
    Keywords: protein crystallography ; four helix bundle ; iron ; macromolecular assembly ; regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a=b=c= 182 ± 0.5 Å. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 Å, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 Å. Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu-57, -58, -59, and -61 are all replaced by Ala. The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541-544, 1991) as the model. The crystallographic R factor for the 6-2.2 Å shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 Å. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function. © 1994 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340180204
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    Distribution function implied dynamics versus residence times and correlations: Solvation shells of myoglobin (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 18 (1994), S. 148-160 
    Details
    ISSN: 0887-3585
    Keywords: myoglobin ; solvation ; dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The dynamics of water at the protein-solvent interface is investigated through the analysis of a molecular dynamics simulation of metmyoglobin in explicit aqueous environment. Distribution implied dynamics, harmonic and quasiharmonic, are compared with the simulated macroscopic dynamics. The distinction between distinguishable solvent molecules and hydration sites developed in the previous paper is used. The simulated hydration region within 7 Å from the protein surface is analyzed using a set of 551 hydration sites characterized by occupancy weights and temperature B-factors determined from the simulation trajectory. The precision of the isotropic harmonic and anisotropic harmonic models for the description of proximal solvent fluctuations is examined. Residence times and dipole reorientation times of water around the protein surface are compared with NMR and ESR results. A correlation between diffraction experiment quantities such as the occupancy weights and temperature factors and the residence and correlation times resulting from magnetic resonance experiments is found via comparison with simulation. © 1994 John Wiley & Sons, Inc.
    Additional Material: 16 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340180207
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    Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 18 (1994), S. 198-200 
    Details
    ISSN: 0887-3585
    Keywords: catalytic antibody ; chorismate mutase ; crystallization ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340180211
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    Structural analysis of two crystal forms of lentil lectin at 1.8 Å resolution (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 330-346 
    Details
    ISSN: 0887-3585
    Keywords: lentil lectin ; legume lectin ; lectin ; side chain clusters ; sugar-protein interactions ; phosphate binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structures of two crystal forms of lentil lectin are determined and refined at high resolution. Orthorhombic lentil lectin is refined at 1.80 Å resolution to anR-factor of 0.184 and monoclinic lentil lectin at 1.75 Å resolution to anR-factor of 0.175. These two structures are compared to each other and to the other available legume lectin structures. The monosaccharide binding pocket of each lectin monomer contains a tightly bound phosphate ion. This phosphate makes hydrogen bonding contacts with Asp-81β, Gly-99β, and Asn-125β, three residues that are highly conserved in most of the known legume lectin sequences and essential for monosaccharide recognition in all legume lectin crystal structures described thus far. A detailed analysis of the composition and properties of the hydrophobic contact network and hydrophobic nuclei in lentil lectin is presented. Contact map calculations reveal that dense clusters of nonpolar as well as polar side chains playa major role in secondary structure packing. This is illustrated by a large cluster of 24 mainly hydrophobic amino acids that is responsible for the majority of packing interactions between the two β-sheets. Another series of four smaller and less hydrophobic clusters is found to mediate the packing of a number of loop structures upon the front sheet. A very dense, but not very conserved cluster is found to stabilize the transition metal binding site. The highly conserved and invariant nonpolar residues are distributed asymmetrically over the protein. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340200406
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    Helix packing in proteins: Prediction and energetic analysis of dimeric, trimeric, and tetrameric GCN4 coiled coil structures (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 105-123 
    Details
    ISSN: 0887-3585
    Keywords: structure prediction ; helix to helix packing ; coiled coils ; leucine zippers ; heptad repeats ; molecular dynamics ; simulated annealing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A simulated annealing method for atomic resolution structure prediction of α-helical coiled coil proteins is described which draws upon knowledge of the oligomerization state, the helix directionality, and the properties of heptad repeat sequences. Unknown structural parameters, such as the coiled coil twist angle and the side chain conformations, are heavily sampled while allowing for flexibility in the helix backbone geometry. Structures of the wild-type GCN4 dimer [O'Shea et al., Science 254:539-544, 1991] and a mutant tetramer [Harbury et al., Science 292:1401-1407, 1993] have been generated and compared with the X-ray crystal structures. The wild-type dimer model has a root mean square coordinate deviation from the crystal structure of 0.73 Å for nonhydrogen atoms in the dimerization interface. Structures of a mutant dimer and a mutant trimer have been predicted. Packing energetics were analyzed for core leucine and isoleucine side chains in dimeric and tetrameric coiled coils. Strong packing preferences were found in the dimers but not in the tetramers. Thus, packing in the dimer may be responsible for the switch from a two-stranded to a four-stranded coiled coil caused by the GCN4 leucine zipper mutations. © 1994 Wiley-Liss, Inc.
    Additional Material: 14 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340200202
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    A special-purpose computer for molecular dynamics: GRAPE-2A (1994)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 20 (1994), S. 139-148 
    Details
    ISSN: 0887-3585
    Keywords: hardware ; molecular dynamics ; simulation ; special-purpose computer ; supercomputing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulations have been extensively used in research of proteins. Since these simulations are quite computer intensive, their acceleration is of main interest of the research. In molecular dynamics simulations, almost all computing time is consumed in calculating the forces between particles, e.g., Coulomb and van der Waals forces. We have designed and built GRAPE-2A (GRAvity PipE 2A), a special-purpose computer for use in simulations of classical many-body systems. GRAPE-2A calculates forces exerted on a particle from the other particles. GRAPE-2A can calculate force of an arbitrary functional form of a central force. The host computer, which is connected to GRAPE-2A through the VME bus, performs other calculations such as time integration. The peak speed of GRAPE-2A is 180 Mflops. We can also stimulate systems with periodic boundary conditions by the Ewald method, using GRAPE-2A and another special-purpose computer, WINE (Wave space INtegrator for the Ewald method). © 1994 Wiley-Liss, Inc.
    Additional Material: 10 Ill.
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    Occurrence of fibers and their association with talin in the cleavage furrows of PtK2 cells (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 26-40 
    Details
    ISSN: 0886-1544
    Keywords: cleavage furrows ; cytokinesis ; actin ; phalloidin ; myosin ; filamin ; talin ; attachment plaques ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley-Liss, Inc.
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    URL: http://dx.doi.org/10.1002/cm.970270104
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    Role of MAPs and motors in the bundling and shimmering of native microtubules from insect ovarioles (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 69-78 
    Details
    ISSN: 0886-1544
    Keywords: kinesin ; dynein ; MAP-motor interactions ; microtubule arrays ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark-field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent-extracted and reactivated. We have studied the nucleotide-specificity and the drug-sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270108
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    Role of microtubules in random cell migration: Stabilization of cell polarity (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 88-96 
    Details
    ISSN: 0886-1544
    Keywords: cell movement ; speed ; persistence time ; colcemid ; alveolar macrophage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of microtubules in random cell migration was investigated using time-lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long-term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270110
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270301
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    Cellular infrared detector appears to be contained in the centrosome (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 262-271 
    Details
    ISSN: 0886-1544
    Keywords: 3T3 cells ; cell motility ; infrared ; phototaxis ; centrosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 μm away from the cell body if the particles were irradiated by an infrared beam in the range of 700-900 nm [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht-Buehler, 1981: Cell Motil. Cytoskeleton 1:237-245]. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270307
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    Role of cAMP in the reactivation of demembranated ram spermatozoa (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 206-218 
    Details
    ISSN: 0886-1544
    Keywords: sperm motility ; sperm maturation ; flagella ; protein kinases ; protein kinase inhibitor ; cGMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained 〉90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270303
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    Announcement (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 286-286 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270309
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  • 73
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    Physical model of axonemal splitting (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 287-298 
    Details
    ISSN: 0886-1544
    Keywords: cilium ; flagellum ; motility ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A physical model developed to explain microtubule sliding patterns in the trypsintreated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the dublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward ( + ) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N-1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one-half of the axoneme are predicted to be active, in a manner consistent with the switch-point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms. © 1994 Wiley-Liss, Inc.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270402
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    Cell motility and the cytoskeleton video supplement 4 (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 361-372 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 16 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270408
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280301
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  • 76
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    Microtubule-associated protein function: Lessons from expression in spodoptera frugiperda cells (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 195-198 
    Details
    ISSN: 0886-1544
    Keywords: baculovirus ; tau ; MAP2 ; neuronal cytoskeleton ; growth cones ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The phenotypes induced by the expression of neuronal microtubule-associated proteins (MAPs) in Sf9 cells have provided data on the in situ function of these proteins. Both MAP2 and tau can induce long processes in Sf9 cells, and the processes contain bundles of microtubules. In both cases the microtubules are aligned with their plus ends distal. Tau expression usually induces a single process that is unbranched and of uniform caliber. Processes can form even when the cells are grown in suspension. Microtubules do not extend all the way to the tip; instead the terminal region contains an actin-rich meshwork. Taxol treatment of Sf9 cells also induces the assembly of microtubules into bundles but does not induce process formation in Sf9 cells. Therefore the in vitro properties of tau as a molecule capable of assembling, stabilizing, and bundling microtubules do not fully account for the in vivo ability of tau alone to transduce microtubule assembly into a change in cell shape. The morphological features of the processes induced by MAP2 differ in highly informative ways. © 1994 Wiley-Liss, Inc.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280302
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    Movement of axoplasmic organelles on actin filaments from skeletal muscle (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 231-242 
    Details
    ISSN: 0886-1544
    Keywords: squid axoplasm ; organelle movement ; calmodulin ; actin filaments ; axonal transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280306
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    A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 265-277 
    Details
    ISSN: 0886-1544
    Keywords: intermediate filament proteins ; vimentin ; domain function ; filament assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280309
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    Dynamic instability of microtubules from cold-living fishes (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 327-332 
    Details
    ISSN: 0886-1544
    Keywords: tubulin ; isoforms ; Atlantic cod ; Antarctic fish ; evolutionary aspects ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynamic instability of microtubules free of microtubule-associated proteins from two genera of cold-living fishes was measured, by means of video-enhanced differential interference-contrast microscopy, at temperatures near those of their habitats. Brain microtubules were isolated from the boreal Atlantic cod (Gadus morhua; habitat temperature ∽ 2-15°C) and from two austral Antarctic rockcods (Notothenia gibberifrons and N. coriiceps neglecta; habitat temperature ∽ -1.8 to + 2°C). Critical concentrations for polymerization of the fish tubulins were in the neighborhood of 1 mg/ml, consistent with high interdimer affinities. Rates of elongation and frequencies of growth-to-shortening transitions (“catastrophes”) for fish microtubules were significantly smaller than those for mammalian microtubules. Slow dynamics is therefore an intrinsic property of these fish tubulins, presumably reflecting their adaptation to low temperatures. Two-dimensional electrophoresis showed striking differences between the isoform compositions of the cod and the rockcod tubulins, which suggests that the cold-adapted microtubule phenotypes of northern and southern fishes may have arisen independently. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280406
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970290101
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  • 81
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    Novel touch-induced, Ca2+-dependent phobic response in a flagellate green alga (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 97-109 
    Details
    ISSN: 0886-1544
    Keywords: calcium ; flagellar movement ; mechanotransduction ; mechanoshock response ; Spermatozopsis similis ; video analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The biflagellate green alga Spermatozopsis similis exhibits a remarkable avoidance reaction in addition to the photophobic or stop response characteristic of such algae. S. similis normally swims forward with its anteriorly attached flagella directed posteriorly and propagating sine-like waves from base to tip. Upon contact with surfaces or other cells, S. similis responds with rapid backward swimming, covering distances of up to 50 μm in 140 to 220 msec. This reaction, which we term the mechanoshock response, also can be triggered by vigorous mechanical stimulation, but not by physiological light intensities. It consists of 3 phases: (1) a rapid acceleration phase with average duration of 31 msec; (2) a phase of about 66 msec with constant high speed (maximal velocities of 〉 600 μm·sec-1) or slow deceleration; and (3) a deceleration phase of ∼ 83 msec, followed by a stop or short period of circling. The cells then resume forward swimming in a random direction. Prior to the mechanoshock response the flagella rapidly are brought together into a close parallel configuration extending anteriorly of the cell body. They then appear to propel the cell by undulatory beating, while the cell describes a pronounced helical path. Small decreases in the extracellular Ca2+ concentration, as well as low concentrations of Ba2+, strongly suppress the probability of this phobic reaction. We conclude that this mechanoshock response involves large Ca2+ influxes, probably mediated by mechanosensitive and/or stretch-activated ion-channel(s). © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290202
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    Kinesin-related polypeptide is associated with vesicles from Corylus avellana pollen (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 155-166 
    Details
    ISSN: 0886-1544
    Keywords: Golgi vesicles ; pollen ; pollen tube ; microtubules ; kinesin-related protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A 100-kDa polypeptide with microtubule-interacting properties was identified in a Golgi vesicle-enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132-137] localized the polypeptide on the external surface of membrane-bounded organelles. Some 100-kDa-containing vesicles co-pelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule-interacting protein, salt-extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2-fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100-kDa polypeptide is part of a molecular complex showing properties of the kinesin class. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290207
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  • 83
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    Nanometer scale vibration in mutant axonemes of Chlamydomonas (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 177-185 
    Details
    ISSN: 0886-1544
    Keywords: flagella ; Chalamydomonas ; mutant ; high-frequency vibration ; nanometer scale measurement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Flagellar axonemes of sea urchin sperm display high frequency (200-400 Hz) vibration with nanometer scale amplitudes in the presence of ATP [Kamimura and Kamiya, 1992: J. Cell Biol. 116:1443-1454]. To investigate how various axonemal components affect the vibration, we examined vibration in wild-type and mutant axonemes of Chlamydomonas. At 1 mM ATP, wild-type axonemes underwent vibration at 100-650 Hz with amplitudes of 4-40 nm. This vibration was similar to, but less regular than, that in sea urchin sperm. Axonemes of the mutants ida1 and ida4 lacking part of the inner arm dynein underwent vibrations indistinguishable from that of wild-type. The mutant oda1 lacking the entire outer arm underwent vibration at about half the wild-type frequency. Unexpectedly, the paralyzed mutants pf18 lacking the central pair and pf14 lacking the radial spokes displayed vibration with significantly higher frequencies and smaller amplitudes than those in the wild-type vibration. These results indicate that the high-frequency vibration is common to many kinds of mutant axonemes that lack various axonemal substructures, but that its manner is sensitive to the presence of outer arm dynein and the central pair/radial spoke system. Simultaneous measurements of amplitude and frequency in wild-type and mutant axonemes suggest that the velocity of microtubule sliding in vibrating axonemes is lower than the velocity of sliding under load-free conditions. The velocity is particularly low in pf18. A possible mechanism is proposed to explain the lower sliding velocity and vibration amplitude in the pf18 axoneme, based on an assumption that central pair/radial spoke system may work to regulate the switching of two antagonizing forces within the axoneme. © 1994 Wiley-Liss, Inc.
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    URL: http://dx.doi.org/10.1002/cm.970290209
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    22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 215-224 
    Details
    ISSN: 0886-1544
    Keywords: cytoplasmic dynein ; Paramecium ; monoclonal antibody ; 12S dynein ; microtubule gliding ; Km and Vmax ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290304
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970290301
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    Phosphoprotein phosphatase 1 (PP1) is a component of the isolated sea urchin mitotic apparatus (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 280-290 
    Details
    ISSN: 0886-1544
    Keywords: mitosis ; phosphorylation ; protein phosphatase ; okadaic acid ; mitotic apparatus ; sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein component of isolated mitotic apparatus having a relative molecular mass of 62,000 (p62) is a substrate of a calcium/calmodulin dependent protein kinase, and the phosphorylation of p62 in vitro correlates directly with microtubule disassembly. In vivo experiments have determined the phosphorylation of p62 increases after fertilization; maximum incorporation of phosphate occurs during late metaphase/early anaphase and decreases thereafter. Because the level of p62 is constant throughout the cell cycle [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-54] the decrease in phosphorylation of p62 observed after anaphase onset is most likely due to the action of a phosphatase. By examination of the relative amount of phosphorylated p62 which remained radiolabeled as a function of time using a standard in vitro phosphorylation assay, the activity of a phosphoprotein phosphatase capable of dephosphorylating p62 in the isolated mitotic apparatus was observed. To characterize the p62 phosphatase, okadaic acid and calyculin A were used to inhibit the dephosphorylation of p62 in vitro. It was found that specific concentrations of okadaic acid (50-500 nM) and of calyculin A (10-100 nM) were effective at inhibiting the dephosphorylation of p62 in vitro. Lower concentrations of either inhibitor had a negligible effect on dephosphorylation of p62. These data indicate the presence of phosphoprotein phosphatase type 1 activity associated with mitotic apparatus isolated from sea urchin embryos using the procedures described here. The implications of these findings relative to our understanding of the regulation of mitosis and cytokinesis are discussed. © 1994 Wiley-Liss, Inc.
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    URL: http://dx.doi.org/10.1002/cm.970290311
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    Gain setting in Chlamydomonas reinhardtii: Mechanism of phototaxis and the role of the photophobic response (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 225-230 
    Details
    ISSN: 0886-1544
    Keywords: Weber's Law ; retinal ; retinal analogs ; photoreception ; alga ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The unicellular green alga Chlamydomonas reinhardtii maintains sensitivity of its phototaxis response (alignment of swimming direction along the axis of a light beam) over several orders of magnitude of light intensities. It is widely accepted that the rotation of the swimming cell provides temporal comparisons of light intensities via periodic contrast generated by its asymmetrically positioned refractile eyespot organelle. The cells also exhibit a second behavioral response to light called the photophobic (or stop) response, which is a brief cessation of swimming caused by a temporal change in light intensity. The cells are desensitized to photophobic stimuli by light exposure. Through comparative measurements of both responses, we explain the behavioral basis of the large dynamic range of phototaxis in terms of precise desensitization of the photophobic response. The basis of the explanation is that the flagellar beat changes which cause phototactic orientation are the residual of the photophobic response after desensitization (i.e., “mini-photophobic” reactions which cause brief reorienting motions without a full stop). This interpretation predicts quantitatively the dependence of the extent of desensitization on light intensity and the dependence of onset and maintenance of phototaxis on extent of desensitization. These predictions are tested and confirmed in this report. © 1994 Wiley-Liss, Inc.
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    URL: http://dx.doi.org/10.1002/cm.970290305
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    Switching of the dominant calcium sequestering protein during skeletal muscle differentiation (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 259-270 
    Details
    ISSN: 0886-1544
    Keywords: calsequestrin ; calreticulin ; sarcoplasmic reticulum ; skeletal muscle ; myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A major Ca2+-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca2+-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, antidesmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation. © 1994 Wiley-Liss, Inc.
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    URL: http://dx.doi.org/10.1002/cm.970290309
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    Interactions among endoplasmic reticulum, microtubules, and retrograde movements of the cell surface (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 291-300 
    Details
    ISSN: 0886-1544
    Keywords: endoplasmic reticulum ; DiOC6(3) ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double-labeling with DiOC6(3) and anti-tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at ∼0.3 μm/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol-stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. © 1994 Wiley-Liss, Inc.This Article is a US Government work and, as such, is in the public domain in the United States of America.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970290402
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  • 90
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    β-actin specific monoclonal antibody (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 108-116 
    Details
    ISSN: 0886-1544
    Keywords: smooth muscle ; fibroblasts ; lamellipodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using a synthetic peptide mimicking the NH2-terminus of β-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the β isoform, by its exclusive recognition of the synthetic β-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the β-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270203
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  • 91
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    Axonemes paralyzed by the presence of dyneins unable to use ribose-modified ATP (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 161-168 
    Details
    ISSN: 0886-1544
    Keywords: fluorescent nucleotide analogs ; methylanthraniloyl ATP ; anthraniloyl ATP ; Chlamydomonas ; axonemal mutants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Substrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose-modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild-type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270207
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  • 92
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    ATP-induced sliding of microtubules on tracks of 22S dynein molecules aligned with the same polarity (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 180-191 
    Details
    ISSN: 0886-1544
    Keywords: sliding movement ; 22S dynein ; Tetrahymena cilia ; dynein-track ; singlet microtubule ; ATP ; polarity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule-dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule-dynein complexes (α-and β-complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)-induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP-induced extrusion of single microtubules. The α-complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β-complex, prepared by adding dynein bound to DEAE-tubulin to pre-assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β-complex extruded at 12 μm/second following the addition of ATP. Dark-field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein-track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark-field microscope, the dynein-track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β-complex under electron microscope. The higher sliding velocity of the microtubules on these dynein-tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270209
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  • 93
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    Antisense MAP-2 oligonucleotides induce changes in microtubule assembly and neuritic elongation in pre-existing neurites of rat cortical neurons (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 234-247 
    Details
    ISSN: 0886-1544
    Keywords: microtubule-associated protein 2 ; neurons ; microtubule-associated proteins ; cytoskeleton ; dendrites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated protein 2 (MAP-2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP-2, two MAP-2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP-2 cDNA were added to rat primary embryonic day 17-18 (E17-18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP-2 AS oligonucleotides resulted in decreased MAP-2 and reduction in the number of neuritic processes relative to the control or MAP-2 sense-treated cultures. By immunostaining and light microscopy the AS-treated neurons appeared smaller, more rounded, and less intensely stained for MAP-2 than the untreated or the MAP-2 sense-treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS-treated cultures were observed. We conclude that MAP-2 continues to be required for microtubule spacing and stability within neurites once they have formed. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270305
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  • 94
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270401
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  • 95
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    Functional analysis of a cardiac myosin rod in Dictyostelium discoideum (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 313-326 
    Details
    ISSN: 0886-1544
    Keywords: myosin II ; cardiac ; sarcomeric ; cytoplasmic myosin ; LMM ; Dd ; heterologous expression ; ConA ; receptor capping ; aggregation ; filament assembly region ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cyto-kinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1-11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677-688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat β cardiac LMM was transfected into both wild-type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild-type and Dd mhc null cells. Although null cells expressing chimeric mhc at ∼10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270404
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  • 96
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    Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335 (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 337-349 
    Details
    ISSN: 0886-1544
    Keywords: microtubules ; glutamylation ; Paramecium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubular networks are extensively developped in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82-85; Eddé et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules.Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies. © 1994 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970270406
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  • 97
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    Actin cytoskeleton and motility in rat sarcoma cell populations with different metastatic potential (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 25-33 
    Details
    ISSN: 0886-1544
    Keywords: actin structures ; vinculin ; focal contacts ; spontaneous metastasis ; extracellular pH ; cell motility ; malignancy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three-dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three-dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 μm2 to 0.4 μm2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time-lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three-dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro. © 1994 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280103
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  • 98
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    Masthead (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280201
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  • 99
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    Purification of microtubule proteins from Xenopus egg extracts: Identification of a 230K MAP4-like protein (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 108-118 
    Details
    ISSN: 0886-1544
    Keywords: Xenopus MAPs ; microtubule cycling ; 230 kDa heat-stable MAP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe the purification of microtubule proteins from Xenopus egg extracts by temperature-dependent assembly and disassembly in the presence of dimethyl sulfoxide and identify a number of presumptive microtubule-associated proteins (MAPs). One of these proteins has a molecular weight of 230 kDa and is immunologically related to HeLa MAP4. We show that this MAP is heat stable and phosphorylated, and that it promotes elongation of microtubules from axonemes. © 1994 Wiley-Liss, Inc.
    Additional Material: 11 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280203
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  • 100
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    Orientation, assembly, and stability of microtubule bundles induced by a fragment of tau protein (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 143-154 
    Details
    ISSN: 0886-1544
    Keywords: microtubule-associated proteins ; microtubule nucleation ; tubulin ; cytoskeleton ; axon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The neuronal microtubule-associated protein tau has been implicated in the development of axonal morphology including the organization of microtubules into a uniformly oriented array of microtubules commonly referred to as “bundle.” Determination of the functional organization of tau has revealed that regions of tau protein which flank the microtubule-binding domain affect the bundling of microtubules in vitro with a microtubule-binding fragment of tau being most effective [Brandt and Lee, 1993: J. Biol. Chem. 268:3414-3419]. In order to study the relation of microtubule bundles that form in vitro to those observed in the axon, we determined the orientation of individual microtubules in bundles and the effects of bundling on microtubule assembly and stability in cell-free assembly reactions. Here we report that bundles induced by a microtubule-binding fragment of tau contain randomly oriented microtubules as determined by using the difference in growth rates at microtubule plus and minus ends. We demonstrate that in vitro bundling increases microtubule growth (about 30%), stabilizes microtubules against dilution- and cold-induced disassembly, and allows microtubule nucleation despite the absence of a tau region which has previously been shown to be required for tau-dependent microtubule nucleation. We conclude that conditions that stabilize microtubules can lead to bundle formation and allow microtubule assembly by a mechanism different from that employed by microtubule-associated proteins. The data also support the view that additional mechanisms besides the action of tau and tubulin exist in order to organize microtubules in the axon. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970280206
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