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31.
Background
Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems.Methodology/Principal Findings
As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state.Conclusions
Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth. 相似文献32.
This article unpacks the history of a 'tribal' region in Central India to show that the current appearance of 'two-dimensionality' or stark opposition between the people and the state is a product of colonial and post-colonial policies rather than a pre-colonial relic. It challenges the idea of 'coercive subordination' as an adequate explanation for kingship in this area, as argued by the late Alfred Gell. Instead, this article uses the same phenomena, annual Dussehra rituals and successive rebellions, to argue for a more dialectical concept of hegemony. It also takes issue with culturalist interpretations of rule, arguing instead for a historically nuanced political economy. 相似文献
33.
BackgroundThe recent increase in the number and severity of cases of nosocomial Clostridium difficile-associated diarrhea (CDAD) has prompted interest in the use of probiotics for the prevention and treatment of this disease. We performed a systematic review of randomized controlled trials to assess the effectiveness of probiotic therapy.MethodsWe searched the PubMed, EMBASE, INAHTA, HEN and Cochrane Collaboration databases to identify trials in which the prevention or treatment of CDAD with probiotic therapy was the primary or secondary outcome. We extracted data on the number of patients randomly assigned to receive probiotic or placebo, the number of patients with CDAD, the type of probiotic, criteria for diagnosing CDAD, persistence of infection after treatment, compliance and adverse effects.ResultsWe identified 4 eligible studies in which prevention (n = 1) or treatment (n = 3) of CDAD was the primary outcome. The benefit of probiotic therapy seen in 2 of the studies was restricted to subgroups characterized by severe CDAD and increased use of vancomycin. The remaining 2 studies were too methodologically flawed for us to draw meaningful conclusions. We also identified 4 trials in which prevention of antibiotic-associated diarrhea with probiotics was the primary outcome and prevention of CDAD a secondary outcome. These studies were limited primarily by too few CDAD cases and provided no evidence of effective prophylaxis. Overall, heterogeneity in choice and dose of probiotic and in criteria for diagnosing CDAD makes it difficult to synthesize information from the 8 studies.InterpretationStudies conducted to date provide insufficient evidence for the routine clinical use of probiotics to prevent or treat CDAD. Better designed and larger studies are needed.Both the incidence and severity of Clostridium difficile-associated diarrhea (CDAD) have increased in hospitals across North America.1,2,3,4 Recent outbreaks in Montreal and Sherbrooke resulted in a 4-fold increase in the number of CDAD cases as well as an increase in the number of attributable deaths.1,5,6,7 CDAD is a debilitating and costly illness, particularly among patients with recurring episodes. Probiotics, or naturally occurring “good bacteria,” have been suggested as a means of both preventing and treating the disease.8,9,10,11 The disturbance of normal gastrointestinal flora, particularly after antibiotic use, is believed to predispose patients to colonization by C. difficile;2 probiotics, by delivering bacteria to the gastrointestinal tract, are believed to restore equilibrium in the altered gastrointestinal flora and thus protect against colonization.8 Probiotics that have been proposed for prevention and treatment of antibiotic-associated diarrhea and CDAD include various bacteria (Bifidobacterium, Lactobacillus GG, L. rhamnosus, L. casei, L. plantarum 299v, Enteroccus faecium [SF68]) and yeasts (Saccharomyces boulardii, S. cerevisiae). They are commonly available as lyophilized capsules or in the form of a fermented drink. We performed a systematic review of randomized controlled trials of probiotic therapy for the prevention and treatment of CDAD. 相似文献
34.
We evaluated the combined effects of food (0.5 × 106, 1.0 × 106 and 2.0 × 106 cells ml−1 of Chlorella vulgaris) and temperature (15, 20 and 25 °C) on life history variables of B. havanaensis. Regardless of Chlorella density there was a steep fall in the survivorship of B. havanaensis at 25 °C. Both food level and temperature affected the fecundity of B. havanaensis. At any given food level, rotifers cultured at 15 °C showed extended but low offspring production. At 25 °C, offspring production was elevated, the duration of egg laying reduced and the fecundity was higher during the latter part of the reproductive period. The effect of food level was generally additive, at any given temperature, and higher densities of Chlorella resulted in higher offspring production. Average lifespan, life expectancy at birth and generation time were 2–3 times longer at 15 °C than at 25 °C. At 20 °C, these remained at intermediate levels. The shortest generation time (about 4 days) was observed at 25 °C. Gross and net reproductive rates and the rate of population increase (r) increased with increasing temperature and generally, at any given temperature, higher algal food levels contributed to higher values in these variables. The r varied from 0.11 to 0.66. The survival patterns and lower rates of reproduction at 15 °C suggest that the winter temperatures (10–15 °C) prevailing in many waterbodies in Mexico City allow this species to sustain throughout the year under natural conditions. 相似文献
35.
Aonghus?J?O'Loughlin Crochan?J?O'Sullivan Nandini?Ravikumar Anne?M?Friel John?T?Elliott John?J?MorrisonEmail author 《Reproductive biology and endocrinology : RB&E》2005,3(1):8
Background
The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro. 相似文献36.
SSS Sarma Lynda Beladjal S Nandini Gerardo Cerón-Martínez Karina Tavera-Briseño 《Aquatic biosystems》2005,1(1):1-7
Background
Freshwater anostracans inhabit ephemeral water bodies in which as the water level decreases due to evaporation the salt concentration increases. Thus, for most anostracans salinity becomes the major stress factor.Results
We tested five concentrations of NaCl (0 to 8 g/l) on the life table demography of Branchipus schaefferi fed Chlorella (alga). Age-specific survivorship curves of male and female B. schaefferi showed nearly a similar pattern in that increased salt concentration resulted in decreased survivorship. The age-specific reproduction (mx) of females showed several peaks of cyst production at 0 and 1 g/l salinity while in treatments containing salt at 4 or 8 g/l, there were fewer peaks. Average lifespan, life expectancy at birth, gross and net reproductive rates, generation time and the rate of population increase were all significantly influenced by the salt concentration in the medium. The highest value of net reproductive rate (970 cysts/female) was in treatments containing 0 g/l of salt, while the lowest was 13 cysts/female at 8 g/l. The rate of population increase (r) varied from 0.52 to 0.32 per day depending on the salt concentration in the medium.Conclusion
The low survival and offspring production of B. schaefferi at higher salinity levels suggests that this species is unlikely to colonize inland saline water bodies. Therefore, the temporary ponds in which it is found, proper conservative measures must be taken to protect this species. 相似文献37.
Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation. 相似文献
38.
Venkatesan BA Mahimainathan L Ghosh-Choudhury N Gorin Y Bhandari B Valente AJ Abboud HE Choudhury GG 《Cellular signalling》2006,18(4):508-518
Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1. 相似文献
39.
Falisse-Poirrier N Ruelle V ElMoualij B Zorzi D Pierard O Heinen E De Pauw E Zorzi W 《Journal of microbiological methods》2006,67(3):593-596
We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines. 相似文献
40.
Chinthamani S Odusanwo O Mondal N Nelson J Neelamegham S Baker OJ 《American journal of physiology. Cell physiology》2012,302(7):C968-C978
Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS). 相似文献