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991.
992.
The prostaglandin transporter "PGT" interacts electrostatically with its anionic substrate, based on inhibition by the disulfonic stilbenes [Chan, B. S. (1998) J. Biol. Chem. 273, 6689-6697], inhibition by the thiol-reactive anion sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570], and the requirement for a negatively charged 1-position carboxyl on the substrate [Itoh, S. (1996) Mol. Pharm. 50, 736-742]. Here we found that modification of positively charged residues on wild-type PGT by arginine- and lysine-specific reagents significantly inhibited transport. We previously found that the binding site of PGT is formed, at least in part, by its membrane-spanning segments [Chan, B. S. (1999) J. Biol. Chem. 274, 25564-25570]. Three charged residues within predicted transmembrane spans (E78, R560, and K613) are conserved in PGT and in related transporters. Substitution of the anionic residue E78 (E78D and E78C) produced an essentially functional transporter, whereas substitution of the cationic residues with neutral residues (R560N and K613Q) resulted in poorly functional transporters. Immunoblotting revealed similar expression levels of wild-type and mutant transporters, and immunostaining indicated correct targeting. Conservative charge substitutions (R560K, K613R, and K613H) resulted in generally functional transporters. In contrast, R560N was nonfunctional, whereas the substrate affinity of K613G decreased greater than 50-fold. Conservative substitutions retaining the charge at position 613 (K613R and K613H) restored the substrate affinity, suggesting a direct role of K613 in substrate binding. Double-neutral mutants E78G/R560C and E78G/K613C were inactive, indicating that these residues are not simply charge-paired. Our results suggest that an arginine at position 560 is critical for maximal substrate translocation, and that a positively charged side chain at position 613 contributes to electrostatic binding of the anionic substrate. 相似文献
993.
Increasingly, genetic studies of tumors of the same histologic diagnosis are elucidating subtypes that are distinct with respect to clinical endpoints such as response to treatment and survival. This raises concerns about the efficiency of using the simple log-rank test for analysis of treatment effect on survival in studies of possibly heterogeneous tumors. Furthermore, such studies, designed under the assumption of homogeneity, may be severely underpowered. We derive analytic approximations for the asymptotic relative efficiency of the simple log-rank test relative to the optimally weighted log-rank test and for the power of the simple log-rank test when applied to subjects with unobserved heterogeneity, as reflected in a continuous frailty, that may interact with treatment. Numerical studies demonstrate that the simple log-rank test may be quite inefficient if the frailty interacts with treatment. Further, there may be a substantial loss of power in the presence of the frailty with or without an interaction with treatment. 相似文献
994.
Schraw W Li Y McClain MS van der Goot FG Cover TL 《The Journal of biological chemistry》2002,277(37):34642-34650
A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity. 相似文献
995.
Ge X Chen J Li N Lin Y Sun C Cao K 《Journal of biochemistry and molecular biology》2003,36(6):603-607
Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures. 相似文献
996.
Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication 下载免费PDF全文
Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase. 相似文献
997.
Cheng H Gao Q Jiang M Ma Y Ni X Guo L Jin W Cao G Ji C Ying K Xu W Gu S Ma Y Xie Y Mao Y 《The international journal of biochemistry & cell biology》2003,35(2):226-234
Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity. 相似文献
998.
Jun Y Yi L Yong T Jianben L Xiong C Qin Z Jiaxin D Songsheng Q Ziniu Y 《Prikladnaia biokhimiia i mikrobiologiia》2003,39(6):656-660
By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B. thuringiensis growth was studied. Copper has been regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu2+ of different concentration have different effects on B. thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B. thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B. thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml. 相似文献
999.
Distraction osteogenesis in correction of micrognathia accompanying obstructive sleep apnea syndrome 总被引:6,自引:0,他引:6
Wang X Wang XX Liang C Yi B Lin Y Li ZL 《Plastic and reconstructive surgery》2003,112(6):1549-57; discussion 1558-9
To evaluate the effect of distraction osteogenesis in correction of micrognathia accompanying obstructive sleep apnea syndrome, a total of 28 patients with different severities of obstructive sleep apnea syndrome underwent mandibular distraction osteogenesis. A total of 51 distraction devices were placed for bilateral distraction in 23 patients and for unilateral distraction in five patients. The mean age of patients was 21.2 years (range, 3 to 60 years). Eleven patients had micrognathia accompanying obstructive sleep apnea syndrome secondary to bilateral temporomandibular joint ankylosis, and 10 patients had micrognathia accompanying obstructive sleep apnea syndrome secondary to unilateral temporomandibular joint ankylosis. Three patients had developmental micrognathia accompanying obstructive sleep apnea syndrome. The other four patients had micrognathia and concomitant obstructive sleep apnea syndrome induced by trauma, infection, or tumor resection. Each patient had been evaluated preoperatively and postoperatively with cephalometry and polysomnography. Mandible advancement ranged from 9 to 30 mm (average, 20.4 mm) and was successfully achieved after distraction. Fine new bone formed in the distraction gap when the distraction devices were removed 3 to 4 months after distraction was completed. No infection or other complications occurred in any patients. Complete curative effects were achieved in nine severe, six moderate, and eight mild obstructive sleep apnea syndrome patients after distraction, and the other five patients had been improved to the mild level. After distraction was completed, the posterior airway space was increased on average from 4.6 mm to 12.5 mm and the sella-nasion-point B angle was increased on average from 66 degrees to 75 degrees on cephalometric studies. The polysomnographic examination showed that the apnea hypopnea index was lowered on average from 58.0 to 3.15, and the lowest oxygen saturation was increased on average from 77 percent to 90.3 percent after distraction was completed. The follow-up period was 3 to 61 months (average, 18.1 months). The curative effect was stable and no relapse occurred. Therefore, the authors conclude that mandibular distraction osteogenesis is an effective method for correcting micrognathia accompanying obstructive sleep apnea syndrome. Compared with other current routine surgical procedures, it has many advantages, such as low risk, simple manipulation, high curative rate, low relapse rate, and stable result. It is presently the most effective method for the treatment of this difficult and complicated disorder. 相似文献
1000.
Zhu Y Nam J Humara JM Mysore KS Lee LY Cao H Valentine L Li J Kaiser AD Kopecky AL Hwang HH Bhattacharjee S Rao PK Tzfira T Rajagopal J Yi H Veena Yadav BS Crane YM Lin K Larcher Y Gelvin MJ Knue M Ramos C Zhao X Davis SJ Kim SI Ranjith-Kumar CT Choi YJ Hallan VK Chattopadhyay S Sui X Ziemienowicz A Matthysse AG Citovsky V Hohn B Gelvin SB 《Plant physiology》2003,132(2):494-505
Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants. 相似文献