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31.
Because interactions between cisplatin and plasma proteins contribute to drug efficacy and side effects, it is important to understand both the binding sites of cisplatin on the proteins and the formation of protein–cisplatin adducts. Previous results suggest that cisplatin preferentially binds to residues on the protein surface. The present work employed electrospray ionization mass spectrometry (MS) to identify such sites on both native and denatured ubiquitin (Ub). Fourier transform (FT) MS and tandem MS (MS/MS and MS3) enable analysis of Ub–cisplatin adduct digests to locate specific cisplatin binding sites. Results indicate that there are three such binding sites, i.e., M1, T12 and T14, and D32, on native Ub. The intensity of the relevant peaks in the FT-MS spectrum of the native Ub adduct digest demonstrates that residues T12 and T14 comprise the primary cisplatin binding site under the native conditions rather than residue M1 as reported in previous research studies. It is found in the present work, however, that M1 is the primary binding site on denatured Ub. Comparison of cisplatin binding sites on native and denatured Ub in this research demonstrates that the conformation of a protein significantly influences the preference of cisplatin for specific binding sites. 相似文献
32.
Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16 总被引:1,自引:0,他引:1
The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK
d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site. 相似文献
33.
Male Siberian hamsters (Phodopus sungorus) housed in long days (LD), but not short days (SD) release luteinizing hormone (LH) when exposed to females. This study examined whether this response is specific to a female and identifies the source of a stimulus that induces LH release. Serum concentrations of LH, testosterone (T), follicle stimulating hormone (FSH), and cortisol were examined in all experiments. T concentrations mirrored the LH response; FSH and cortisol were unchanged in response to all stimuli. Exposure to an LD female, irrespective of her reproductive status, but not an SD female, elicited LH release. Exposure to another male did not trigger LH release. Males released LH when allowed physical contact with an anesthetized female, but not when separated from a normally active female, suggesting that tactile or nonvolatile chemosensory stimuli elicit LH release. Urine and secretions collected from the vagina as well as oral, midventral, perineal, and rectal glands, elicited marked behavioral responses in male P. sungorus. Despite these behavioral responses, only feces from females elicited LH release in males. Males released LH in response to feces extracted from the rectum and to cotton swabs that had been rubbed against the rectal mucosa, suggesting that a component of rectal secretions may trigger LH release in male Siberian hamsters. Taken together, these data and previous data from our laboratory indicate that both the production of and the response to a pheromone that triggers the selective release of LH is regulated by day length. 相似文献
34.
35.
Temporary all‐male social groups are formed in a number of animal species. We examined 34 years of data collected from 36 male Thornicroft's giraffe in the Luangwa Valley, Zambia, to test a set of predictions related to five possible functions of all‐male herds (predator protection, practicing aggressive skills, prolonging life, nutritional demands and resource learning). We found that all‐male herds were significantly smaller than mixed‐sex herds, usually contained a mature bull, and were not dependent upon season or habitat. Dyadic associations between males in single sex herds were quite weak, with <25% of potential male dyads sighted together in an all‐male herd. Our data are best explained as a resource learning strategy adopted by males to obtain more extensive knowledge about the habitat, including both food and female distribution. However, other benefits in the form of predator protection, dietary intake and sharpening competitive skills for future contests over estrous females also seem to mediate formation of giraffe all‐male groups. We conclude that the primary advantage of roaming in all‐male herds changes during the life history of males. 相似文献
36.
Background
Saccharomyces cerevisiae has been associated with human life for millennia in the brewery and bakery. Recently it has been recognized as an emerging opportunistic pathogen. To study the evolutionary history of S. cerevisiae, the origin of clinical isolates and the importance of a virulence-associated trait, population genetics and phenotypic assays have been applied to an ecologically diverse set of 103 strains isolated from clinics, breweries, vineyards, fruits, soil, commercial supplements and insect guts.Methodology/Principal Findings
DNA sequence data from five nuclear DNA loci were analyzed for population structure and haplotype distribution. Additionally, all strains were tested for survival of oxidative stress, a trait associated with microbial pathogenicity. DNA sequence analyses identified three genetic subgroups within the recombining S. cerevisiae strains that are associated with ecology, geography and virulence. Shared alleles suggest that the clinical isolates contain genetic contribution from the fruit isolates. Clinical and fruit isolates exhibit high levels of recombination, unlike the genetically homogenous soil isolates in which no recombination was detected. However, clinical and soil isolates were more resistant to oxidative stress than any other population, suggesting a correlation between survival in oxidative stress and yeast pathogenicity.Conclusions/Significance
Population genetic analyses of S. cerevisiae delineated three distinct groups, comprising primarily the (i) human-associated brewery and vineyard strains, (ii) clinical and fruit isolates (iii) and wild soil isolates from eastern U.S. The interactions between S. cerevisiae and humans potentiate yeast evolution and the development of genetically, ecologically and geographically divergent groups. 相似文献37.
Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term and prolonged starvation in organisms, respectively. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on this process. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the p38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA. 相似文献
38.
Bajaj A Connelly SM Gehret AU Naider F Dumont ME 《Biochimica et biophysica acta》2007,1773(6):707-717
The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling. 相似文献
39.
Fred Stutzenberger 《Applied microbiology and biotechnology》1988,28(4-5):387-393
Summary Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by Thermomonospora curvata and as a substrate for enzymatic conversion to soluble sugars. In shake cultures grown at 53°C, pH 8, maximal cellulase (50 endoglucanase IU and 0.7 filter paper IU/ml) was attained at a PELF concentration of 1.3% (w/v) in a vitamin-mineral salts medium buffered with 0.05 M concentrations of phosphate and N-2-hydroxyethylpiperazine-N-2-ethanesulfonate. Multiple endoglucanase enzymes were produced. The major form had an apparent molecular weight of 22 000 and K
m
values of 0.23% and 0.56% for carboxymethylcellulose and PELF respectively. NaOH treatment of PELF increased its susceptibility to enzymatic hydrolysis. During enzymatic hydrolysis of NaOH-treated PELF (60°C, pH 6.1, 24h) two-thirds of total fibre carbohydrate was solubilized as cellobiose, melibiose, cellopentaose, cellotetraose, xylose and glucose in descending order of concentration. 相似文献
40.
Moreland JG Davis AP Matsuda JJ Hook JS Bailey G Nauseef WM Lamb FS 《The Journal of biological chemistry》2007,282(47):33958-33967
Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes. 相似文献