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1.
The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation 总被引:48,自引:38,他引:10
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The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro. 相似文献
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Slow response of soil organic matter to the reduction in atmospheric nitrogen deposition in a Norway spruce forest 总被引:1,自引:0,他引:1
NICOLE DÖRR KLAUS KAISER ROBERT MIKUTTA GEORG GUGGENBERGER 《Global Change Biology》2010,16(11):2990-3003
Global nitrogen (N) deposition rates in terrestrial environments have quadrupled since preindustrial times, causing structural and functional changes of ecosystems. Different emission reduction policies were therefore devised. The aim of our study was to investigate if, and over what timescale, processes of soil organic matter (OM) transformation respond to a decline in atmospheric N deposition. A N‐saturated spruce forest (current N deposition: 34 kg ha?1 yr?1; critical N load: 14 kg ha?1 yr?1), where N deposition has been reduced to 11.5 kg ha?1 yr?1 since 1991, was studied. Besides organic C and organic and inorganic N, noncellulosic carbohydrates, amino sugars and amino acids were determined. A decline in organic N in litter indicated initial effects at plant level. However, there were no changes in biomarkers upon the reduction in N deposition. In addition, inorganic N was not affected by reduced N deposition. The results showed that OM cycling and transformation processes have not responded so far. It was concluded that no direct N deposition effects have occurred due to the large amount of stored organic N, which seems to compensate for the reduction in deposited N. Obviously, the time span of atmospheric N reduction (about 14.5 years) is too short compared with the mean turnover time of litter to cause indirect effects on the composition of organic C and N compounds. It is assumed that ecological processes, such as microbial decomposition or recycling of organic N and C, react slowly, but may start within the next decade with the incorporation of the new litter. 相似文献
3.
Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage,cAMP, and calyculin A 总被引:1,自引:0,他引:1
Pedersen SF King SA Rigor RR Zhuang Z Warren JM Cala PM 《American journal of physiology. Cell physiology》2003,284(6):C1561-C1576
In this report, wedescribe the cloning, cellular localization, and functionalcharacteristics of Na+/H+ exchanger 1 (NHE1)from red blood cells of the winter flounder Pseudopleuronectesamericanus (paNHE1). The paNHE1 protein localizes primarily to themarginal band and exhibits a 74% similarity to the trout -NHE, and65% to the human NHE1 (hNHE1). Functionally, paNHE1 sharescharacteristics of both -NHE and hNHE1 in that it is activated bothby manipulations that increase cAMP and by cell shrinkage,respectively. In accordance, the paNHE1 protein exhibits both proteinkinase A consensus sites as in -NHE and a region of high homology tothat required for shrinkage-dependent activation of hNHE1. Aftershrinkage-dependent activation of paNHE1 and resulting activation of aCl/HCO exchanger, their paralleloperation results in net uptake of NaCl and osmotically obliged water.Activation of paNHE1 by cAMP is at least additive to that elicited byosmotic shrinkage, suggesting that these stimuli regulate paNHE1 bydistinct mechanisms. Finally, exposure to the serine/threoninephosphatase inhibitor calyculin A potently activates paNHE1, and thisactivation is also additive to that induced by shrinkage or cAMP. 相似文献
4.
William A Cafruny Richard G Duman Grace HW Wong Suleman Said Pam Ward-Demo Raymond RR Rowland Eric A Nelson 《Virology journal》2006,3(1):1-17
Background
Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.Results
Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.Conclusion
The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation. 相似文献5.
Rigor RR Beard RS Litovka OP Yuan SY 《American journal of physiology. Cell physiology》2012,302(10):C1513-C1522
Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor G?6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by G?6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions. 相似文献
6.
Kiessoun Konaté Jacques Fran?ois Mavoungou Alexis Nicaise Lepengué Ra?ssa RR Aworet-Samseny Adama Hilou Alain Souza Mamoudou H Dicko Bertrand M’Batchi 《Annals of clinical microbiology and antimicrobials》2012,11(1):1-12
Background
The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.Method
The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.Results
We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.Conclusion
The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes. 相似文献7.
Ortiz-Acevedo A Rigor RR Maldonado HM Cala PM 《American journal of physiology. Cell physiology》2008,295(5):C1316-C1325
Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control. 相似文献
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Nicholas J Boddicker Angelica Bjorkquist Raymond RR Rowland Joan K Lunney James M Reecy Jack CM Dekkers 《遗传、选种与进化》2014,46(1):18