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  • 1
    ISSN: 0886-1544
    Keywords: maytansine ; vinblastine ; diphenylpyridazone ; colchicine ; taxol ; tubulin ; microtubule ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0°C and at 37°C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 μM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 2
    ISSN: 1040-452X
    Keywords: Embryonic stem cells ; Chimeras ; Microsatellites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring.The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample.Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice. © 1993 Wiley-Liss, Inc.
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  • 3
    ISSN: 0886-1544
    Keywords: intermediate filaments ; phosphorylation ; sea urchin embryos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 μM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase.To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 μM 6-DMAP and with 200 μCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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  • 4
    ISSN: 0886-1544
    Keywords: peptide antibodies ; protein processing ; axonemes ; microtubule associated proteins ; UV photocleavage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dyneins are multi-subunit enzymes that transduce chemical energy into the mechanical energy that makes cilia and flagella beat and moves organelles towards the minus end of microtubules. The ATPase activity is borne by heavy chains, and recent molecular analysis indicates that dynein heavy chain genes form an ancient multigene family: the similarity between the same isoform of two distantly related species is greater than that between different isoforms of the same species. We have exploited sequence identities between a Paramecium axonemal dynein heavy chain gene cloned in our laboratory and sequences of dynein heavy chains from other species to prepare antibodies against active-site peptides capable of recognizing dynein heavy chains regardless of species or isoform. One of the antibodies is perfectly specific for the larger product of V1 photolysis (HUV1) and thus incorporates a unique property of the hydrolytic ATP binding site of all known dynein heavy chains, the capacity for photocleavage in the presence of micromolar vanadate. Our characterization of these reagents suggests that they will be useful for biochemical and in situ studies of known dyneins as well as identification of potential new members of the family. © 1994 Wiley-Liss, Inc.
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  • 5
    ISSN: 0886-1544
    Keywords: centrosome ; microtubule ; microfilament ; myogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Available data on the molecular composition of the centrosome, the typical microtubule-organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well-characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 6
    ISSN: 0886-1544
    Keywords: microtubule-associated proteins ; cell cycle ; phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat-soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.
    Additional Material: 8 Ill.
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  • 7
    ISSN: 0886-1544
    Keywords: tubulin structure ; microtubule ; antibody ; microtubule poison ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Polyclonal antibodies have been raised against the peptide 28-38 of the β-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all β'-tubulin subunits, except for β-tubulin isotypes, and did not recognize the α-tubulin subunit. The 28-38 region does not play a role in the interaction between the α-and β-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubule associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of β-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the β-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly. © 1992 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 8
    ISSN: 0741-0581
    Keywords: Ultrastructural immunocytochemistry ; Enkephalins ; Choline acetyltransferase ; GABA ; Glutamate ; Cochlea ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to γ-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.
    Additional Material: 21 Ill.
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  • 9
    ISSN: 0741-0581
    Keywords: Epithelial cells ; Transport ; Intestinal cell ; Flounder ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The polymorphism of the endoplasmic reticulum (ER) in epithelial cells with different transport functions such as the enterocyte suggests that the ER may be involved in some way in molecular transport. To further access this possibility, we examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes an annual fast of approximately 6 months' duration, a time during which previous work indicates nutrient carrier number does not change. Fish from June (feeding) and January (8-10 weeks fasted) were sampled. Tissues from the pyloric caeca, foregut, midgut, and hindgut were prepared for electron microscopy using two techniques of staining. Cell height was unaltered in any section, although microvillar length shortened variably. Cellular organization, including position of nuclei, number and distribution of mitochondria, and presence of basolateral membranes, did not change. The ER appeared equally abundant in June and January. However, use of the osmium impregnation technique, which is specific for ER cisternal contents, revealed a change in the impregnation of ER, from a heavily impregnated network in summer to little or no impregnation in winter. These results suggest that a shift in function of the ER had occurred when nutrient transport ceased, and supports a role of the ER in nutrient transport.
    Additional Material: 11 Ill.
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  • 10
    ISSN: 0741-0581
    Keywords: BrdU incorporation ; Cultured cells DNA replication ; Electron microscopy ; EM immunocytochemistry ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minues. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.
    Additional Material: 14 Ill.
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