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121.
Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered. 相似文献
122.
Tang Z Mandel LL Yean SL Lin CX Chen T Yanagida M Lin RJ 《Experimental cell research》2003,283(1):101-115
The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes. 相似文献
123.
Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation. 相似文献
124.
Patterns of divergence in the effects of mating on female reproductive performance in flour beetles 总被引:3,自引:0,他引:3
Nilsson T Fricke C Arnqvist G 《Evolution; international journal of organic evolution》2002,56(1):111-120
Sexual selection can lead to rapid divergence in reproductive characters. Recent studies have indicated that postmating events, such as sperm precedence, may play a key role in speciation. Here, we stress that other components of postmating sexual selection may be involved in the evolution of reproductive isolation. One of these is the reproductive investment made by females after mating (i.e., differential allocation). We performed an experiment designed to assess genetic divergence in the effects of mating on female reproductive performance in flour beetles, Tribolium castaneum. Females were mated to males of three different wild-type genotypes at two different frequencies, in all possible reciprocal combinations. Male genotype affected all aspects of female reproduction, through its effects on female longevity, total offspring production, reproductive rate, mating rate, and fertility. Moreover, male and female genotype interacted in their effects on offspring production and reproductive rate. We use the pattern of these interactions to discuss the evolutionary process of divergence and suggest that the pattern is most consistent with that expected if divergence was driven by sexually antagonistic coevolution. In particular, the fact that females exhibited a relatively weak response to males with which they were coevolved suggests that females have evolved resistance to male gonadotropic signals/stimuli. 相似文献
125.
Enhanced selection for MHC diversity in social tuco-tucos 总被引:2,自引:0,他引:2
To explore the effects of behavior and demography on balancing selection at major histocompatibility complex (MHC) loci, we examined allelic diversity at exon 2 of the MHC class II DQbeta locus in a social and a solitary species of tuco-tuco (Rodentia: Ctenomyidae: Ctenomys), both of which occur in the same valley in southwestern Argentina. By comparing patterns of diversity at this MHC gene to the diversity evident at fifteen microsatellite loci, we demonstrate that balancing selection at the DQbeta locus is enhanced in the social species compared to its solitary congener. These findings have intriguing implications for the role of behavioral and demographic parameters in maintaining diversity at MHC loci. 相似文献
126.
Wessel I Jensen LH Renodon-Corniere A Sorensen TK Nitiss JL Jensen PB Sehested M 《FEBS letters》2002,520(1-3):161-166
Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme. 相似文献
127.
Iverson TM Luna-Chavez C Croal LR Cecchini G Rees DC 《The Journal of biological chemistry》2002,277(18):16124-16130
The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover. 相似文献
128.
Drosophila topoisomerase (topo) IIIbeta is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5'-end of DNA. We show here that a purified preparation of topo IIIbeta is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 degrees C, maximal cleavage occurs between 23 and 30 degrees C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo IIIbeta possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo IIIbeta cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo IIIbeta may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character. 相似文献
129.
Sumbilla C Lewis D Hammerschmidt T Inesi G 《The Journal of biological chemistry》2002,277(16):13900-13906
Ca(2+) transport by sarcoplasmic reticulum (SR) ATPase occurs with an optimal coupling ratio of 2 Ca(2+) per ATP in pre-steady state. However, slippage of the pump and lower coupling ratios are observed in steady state. Slippage depends on the presence of high Ca(2+) in the lumen of SR vesicles and high nucleotide in the medium. Thereby, Ca(2+) and/or nucleotide-bound phosphoenzyme intermediates accumulate and undergo uncoupled cleavage, before vectorial translocation of bound Ca(2+) in the forward direction of the cycle or before productive reversal to ATP synthesis. Transport efficiency and coupling ratios are improved by reduction of nucleotide concentration in the presence of ATP regenerating systems and/or complexation of luminal Ca(2+) with phosphate or oxalate. Curcumin (1-5 microm) lowers the concentration of phosphate or oxalate required to reduce slippage of the Ca(2+) pump. Thereby, under appropriate conditions, curcumin favors kinetic flow, completion of productive cycles, and improvement of coupling ratios. The findings obtained with isolated SR vesicles suggest that slippage is an important phenomenon under prevailing conditions of muscle fibers in situ. Ca(2+) transport and its slippage can be improved by curcumin in cardiac as well as in skeletal SR, raising the possibility of pharmacological interventions to correct defective Ca(2+) homeostasis. Higher curcumin concentrations (5-30 microm), however, inhibit overall ATPase activity and Ca(2+) transport by interfering with phosphoenzyme formation with ATP or P(i). 相似文献
130.