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21.
A rationale for the design of an inhibitor of tyrosyl kinase 总被引:1,自引:0,他引:1
Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation. 相似文献
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Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes 总被引:2,自引:0,他引:2
Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents. 相似文献
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Cahan SH Graves CJ Brent CS 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2011,181(8):991-999
Parents can influence the phenotypes of their offspring via a number of mechanisms. In harvester ants, whether female progeny
develop into workers or daughter queens is strongly influenced by the age and temperature conditions experienced by their
mother, which is associated with variation in maternal ecdysteroid deposition in fertilized eggs. In many insects, juvenile
hormone (JH) is antagonistic to ecdysteroid release, suggesting that seasonal and age-based variation in maternal JH titers
may explain maternal effects on offspring size and reproductive caste. To test this hypothesis, we artificially increased
maternal JH titers with methoprene, a JH analog, in laboratory colonies of two Pogonomyrmex populations exhibiting genetic caste determination. Increasing maternal JH resulted in a 50% increase in worker body size,
as well as a sharp reduction in total number of progeny reared, but did not alter the genotype of progeny reared to adulthood.
The intergenerational effect of JH manipulation was not mediated by a reduction in ecdysteroid deposition into eggs; instead,
changes in egg size, trophic egg availability or brood/worker ratio may have altered the nutritional environment of developing
larvae. Egg ecdysteroid content was significantly negatively correlated with natural variation in worker body size, however,
suggesting that there are multiple independent routes by which queens can modify offspring phenotypes. 相似文献
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Tariq Ezaz Denis O’Meally Alexander E. Quinn Stephen D. Sarre Arthur Georges Jennifer A. Marshall Graves 《Cytotechnology》2008,58(3):135-139
Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology.
Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction
in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these
cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal
diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian
dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually
unlimited source of material for cytogenetic, evolutionary and genomic studies. 相似文献
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In mammals, birds, snakes and many lizards and fish, sex is determined genetically (either male XY heterogamy or female ZW heterogamy), whereas in alligators, and in many reptiles and turtles, the temperature at which eggs are incubated determines sex. Evidently, different sex-determining systems (and sex chromosome pairs) have evolved independently in different vertebrate lineages. Homology shared by Xs and Ys (and Zs and Ws) within species demonstrates that differentiated sex chromosomes were once homologous, and that the sex-specific non-recombining Y (or W) was progressively degraded. Consequently, genes are left in single copy in the heterogametic sex, which results in an imbalance of the dosage of genes on the sex chromosomes between the sexes, and also relative to the autosomes. Dosage compensation has evolved in diverse species to compensate for these dose differences, with the stringency of compensation apparently differing greatly between lineages, perhaps reflecting the concentration of genes on the original autosome pair that required dosage compensation. We discuss the organization and evolution of amniote sex chromosomes, and hypothesize that dosage insensitivity might predispose an autosome to evolving function as a sex chromosome. 相似文献