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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.”
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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.
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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: 1040-452X
    Keywords: Cycloheximide ; Meiotic maturation ; Emetine ; Puromycin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the control (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP. In a time course experiment, it was determined that CX could prevent FSH-induced maturation if added as late as 3 h after the onset of culture, but the drug was less effective in EGF-treated cultures. It is concluded that a protein with a rapid turnover rate is involved in the spontaneous maturation of mouse oocytes and that de novo protein synthesis is a requirement for hormone induction of GVB in vitro.
    Additional Material: 5 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Developmental Dynamics 196 (1993), S. 174-182 
    ISSN: 1058-8388
    Keywords: Synapse elimination ; Muscle development ; Compartmentalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In adult rats, each neuromuscular compartment of the lateral gastrocnemius muscle (LG) is exclusively innervated by a primary branch of the LG nerve. In neonates, however, a small percentage of LG cells receives inputs from more than one primary nerve branch; these inputs are known as cross-compartmental. Cross-compartmental inputs are normally lost from the medial compartment of LG (LGm) by the 8th postnatal day. To investigate the mechanisms involved in the elimination of cross-compartmental inputs, muscle fibers in the LGm compartment were denervated by cutting the LGm nerve branch in 1-4 day old rat pups and in adult rats. We then assessed the degree of cross-compartmental innervation within the “denervated” compartment using intracellular recordings from neonatal muscle fibers or immunohistochemical staining for nerve cell adhesion molecule (N-CAM) and neurofilament protein in adult muscles. Following LGm axotomy in neonates, cross-compartmental innervation is more extensive than in controls and is present as late as 20 days after birth. Thus, in the absence of “native” LGm axons, neonatal cross-compartmental inputs proliferate by axonal sprouting and the formation of new synapses on vacant LGm fibers. In contrast, axotomized adults do not form new cross-compartmental inputs over the same time period. The differential response of neonates and adults to muscle nerve branch denervation is evidence for the existence of some form of compartment-specific recognition. We propose that compartmental identity either arises or becomes relatively more potent during ontogeny and normally acts selectively to eliminate foreign axons and deter the formation of new cross-compartmental inputs. © 1993 wiley-Liss, Inc.
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