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  • 1
    ISSN: 0886-1544
    Keywords: taxol ; microtubules ; mitosis ; mitotic spindle ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Taxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calciumlabile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 μM Ca2+ was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane-free mitotic spindles, isolated with a calcium-chelating, nonionic detergent buffer, were stored in an EGTA-gylcerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA-buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60-120 min; but in isolated spindles perfused with buffer that contained 100 μM Ca2+, BR decreased by 90% within 2-5 sec. In contrast, spindles that were pretreated for 3 min with 1 μM taxol, or for about 30 sec with 10 μM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 μM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 μM or 10 μM taxol then washout, stability of spindle BR to 100 μM Ca2+ decreased exponentially with a time constant of 30-60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol-stabilized microtubules are not “fixed.”
    Additional Material: 5 Ill.
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  • 2
    ISSN: 0886-1544
    Keywords: dynein ; erythro-9-[3-2-(hydroxynonyl)]adenine (EHNA) ; ATPase ; inhibition ; axoneme ; cytoplasm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein-like ATPase from cell-free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein-like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP-metabolizing enzyme activities, including 13 Mg++-ATPases. We isolate cytoplasmic dynein-like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose-response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose-response characteristics of fourteen other ATP-metabolizing enzyme activities, including seven nondynein Mg++-ATPases, differ quantitatively from those of dynein-like ATPases.In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X-100), we find that dynein-like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X-100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein-like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytoplasmic dynein-like ATPase (s).
    Additional Material: 6 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 2 (1982), S. 121-126 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 2 Ill.
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  • 4
    ISSN: 0886-1544
    Keywords: cardiac muscle ; actin dynamics ; α-actinin ; vinculin ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, α-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells'native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, α-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throug hout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC3[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.
    Additional Material: 9 Ill.
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  • 5
    ISSN: 1040-452X
    Keywords: Meiotic maturation ; Hypoxanthine phosphoribosyltransferase ; MPTF ; 6-MP ; De novo purine synthesis ; Xanthine oxidase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte-cumulus cell complexes. At a concentration of 1 mM, 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6-azauridine and 2,6-bis-(hydroxyamino)-9-β-D-ribofuranosyl-purine had no effect. MPTF and 6-MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine-maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis-arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6-methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine-arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation. © 1993 Wiley-Liss, Inc.
    Additional Material: 10 Ill.
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  • 6
    ISSN: 0886-1544
    Keywords: sea urchin sperm ; motilily ; two dynein ATPases ; force generation ; power output ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.
    Additional Material: 4 Ill.
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  • 7
    ISSN: 0886-1544
    Keywords: mitosis ; calcium ; diacylglycerol ; protein kinase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have treated living, intact stamen hair cells from the spiderwort plant, Tra-descantia virginiana, with 0.5 μg/ml or 60 μg/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-di-octanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to ∼8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 μg/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 μg/ml 1,2-dioctan oylglycerol in late metaphase, ∼26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 μg/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to ∼5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 μg/ml or 60 μg/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added atother times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require 〉55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.
    Additional Material: 8 Ill.
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  • 8
    ISSN: 0886-1544
    Keywords: ATPase ; flagella ; intermediate chains ; vanadate-mediated photolysis ; vertebrate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Outer-arm dynein purified from trout spermatozoa was disrupted by low-ionicstrength dialysis, and the resulting subunits were separated by sucrose density-gradient centrifugation. The intact 19 S dynein, containing the α- an β-heavy chains, intermediate chains (ICs) 1-5 and light chains (LCs) 1-6, yielded several discrete particles: a 17.5 S adenosine triphosphatase (ATPase) composed of the γ-and β-chains ICs 3-5 and LC 1; a 9.5 S complex containing ICs 1 and 2 together with LCs 2, 3, 4, and 6; and a single light chain (LC 5), which sedimented at ∼4 S. In some experiments, ICs 3-5 also separated from the heavy chain complex and were obtained as a distinct subunit. Further dissociation of the 17.5 S particle yielded a 13.1 S ATPase that contained the β-heavy chain and ICs 3-5. The polypeptide compositions of the complexes provide new information on the intermolecular associations that occur within dynein.Substructural features of the trout dynein polypeptides also were examined. The heavy chains were subjected to vanadate-mediated photolysis at the V1 sites by irradiation at 365 nm in the presence of Mg2+, ATP, and vanadate. Fragment pairs of relative molecular mass (Mr) 245,000/185,000 and 245,000/170,000 were obtained from the α- and β-heavy chains, respectively. Photolysis of these molecules at their V2 sites, by irradiation in the presence of vanadate and Mn2+, yielded fragments of Mr 160,000/270,000 and 165,000/250,000, respectively. These values confirm that the α- and β-heavy chains have masses of 430,000 and 415,000 daltons, respectively.Immunological analysis using monoclonal antibodies revealed that one intermediate chain from trout dynein (IC 2) contains epitopes present in two different intermediate chains from Chlamydomonas dynein. This indicates that specific sequences within the dynein intermediate chains have been highly conserved throughout evolution.
    Additional Material: 9 Ill.
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  • 9
    ISSN: 1573-7225
    Keywords: China ; Hawaii ; Japan ; migrant studies ; the Philippines ; thyroid neoplasms ; United States
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: We compared incidence rates of primary cancer of the thyroid among United States-born and foreign-born Chinese, Japanese, and Filipino residents of the US with rates among US-born Whites. Thyroid cancers diagnosed between 1973 and 1986 occurring among individuals 15 to 84 years of age residing in western Washington state, the San Francisco-Oakland (California) area, or the state of Hawaii were included in the analysis. Population estimates by age, gender, ethnicity, and country of birth were obtained for these areas from the US Bureau of the Census. Filipino women born in the Philippines had 3.2 (95 percent confidence interval=2.7–3.8) times the rate of thyroid cancer of US-born White women, while US-born Filipino women were not at any increased risk. Philippine-born Filipino men also had a relatively high rate of thyroid cancer (relative risk [RR]=2.6), more so than US-born Filipino men (RR=1.5). Among Japanese, risk of thyroid cancer varied by birthplace, but the direction of the association differed by gender and by histologic type of cancer. No clear association with birthplace was noted among Chinese men or women. These data suggest that persons residing in one or more regions from which Filipino-Americans migrated have been exposed to environmental influences that have increased their subsequent risk of thyroid cancer.
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  • 10
    ISSN: 1573-7225
    Keywords: Asian-Americans ; China ; Japan ; migrants ; non-Hodgkin's lymphoma ; Philippines ; United States
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: We examined the incidence of non-Hodgkin's lymphoma (NHL) in Chinese, Japanese, and Filipino residents of the United States to obtain further clues about the etiology of the disease. The age, race, and birthplace of residents of Hawaii, San Francisco/Oakland (California), and western Washington who had received a diagnosis of NHL during the period 1973–86 were obtained from population-based cancer registries, and a special tabulation from the 1980 Census was used to estimate the number of person-years at risk for each category of resident. The incidence of NHL in each of the Asian groups examined was 35 to 85 percent that of US-born Whites. However, there was no consistent trend of increasing incidence with increasing generation of residence in any of the groups. In Asian-Americans, the risk of small cell lymphocytic and plasmacytoid lymphoma was 10 to 85 percent that of Whites, although no clear trends of risk with generation of residence in the US were observed. They also were at a reduced risk of follicular lymphoma, and in Chinese and Japanese persons, the risk was lower in first generation than in later generation migrants (Chinese: Asian-born relative risk [RR]=0.11, US-born, RR=0.84; Japanese: Asian-born, RR=0.15, US-born, RR 0.36). The risk of diffuse lymphoma was similar in Chinese-and Japanese-Americans and US-born Whites. We conclude that, with the exception of follicular lymphoma, the basis for the relatively low incidence of NHL in Asian-Americans does not lie in exposures or characteristics that differ between the migrants themselves and their descendants.
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