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An assay method based on the ability of high concentrations of Mg2+ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.  相似文献   
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Abstract Broad host-range RK2-based cosmid vectors ('costramids') are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of 'heterologous' probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene ( gltA ) or Escherichia coli , the gene encoding δ-aminol evulinic acid synthase ( hemA ), and a gene or genes regulating dicarboxylate transport.  相似文献   
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The cerebral cortex is divided into many functionally distinct areas. The emergence of these areas during neural development is dependent on the expression patterns of several genes. Along the anterior-posterior axis, gradients of Fgf8, Emx2, Pax6, Coup-tfi, and Sp8 play a particularly strong role in specifying areal identity. However, our understanding of the regulatory interactions between these genes that lead to their confinement to particular spatial patterns is currently qualitative and incomplete. We therefore used a computational model of the interactions between these five genes to determine which interactions, and combinations of interactions, occur in networks that reproduce the anterior-posterior expression patterns observed experimentally. The model treats expression levels as Boolean, reflecting the qualitative nature of the expression data currently available. We simulated gene expression patterns created by all possible networks containing the five genes of interest. We found that only of these networks were able to reproduce the experimentally observed expression patterns. These networks all lacked certain interactions and combinations of interactions including auto-regulation and inductive loops. Many higher order combinations of interactions also never appeared in networks that satisfied our criteria for good performance. While there was remarkable diversity in the structure of the networks that perform well, an analysis of the probability of each interaction gave an indication of which interactions are most likely to be present in the gene network regulating cortical area development. We found that in general, repressive interactions are much more likely than inductive ones, but that mutually repressive loops are not critical for correct network functioning. Overall, our model illuminates the design principles of the gene network regulating cortical area development, and makes novel predictions that can be tested experimentally.  相似文献   
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Alfalfa (Medicago sativa L. cv. Vernal) nodules were separatedinto host plant fractions and fractions of rhizobial originby differential centrifugation and sedimentation equilibriumcentrifugation. Both NAD- and NADP-linked isocitrate dehydrogenase(70%, 90%) and glutamate dehydrogenase activities (90%, 83%)were located primarily (percent total nodule activity) in thefractions of plant origin and their specific activities werehighest in the fractions of plant origin. More than 50% of thenodules' total activity of both glutamine synthetase and NAD-glutamatesynthase and greater than 90% of the total glutamate oxaloacetatetransaminase activity was located in plant fractions. However,the fractions of rhizobial origin had the highest specific activitiesof glutamine synthetase and glutamate synthase. (Received September 5, 1981; Accepted December 7, 1981)  相似文献   
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We describe a direct analysis for the simultaneous quantitative determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid, utilizing reversed-phase high-performance liquid chromatography with amperometric detection. In addition, a rapid isocratic separation was developed for homovanillic acid in the presence of other endogenous compounds.Twenty-five unselected diagnostic specimens of human lumbar cerebrospinal fluid were extracted with ethyl acetate and subsequently analyzed using the described method. Chromatographic peaks were identified on the basis of retention behavior and ratio of responses at several oxidation potentials.Although our quantitative results correlate well with the literature values, the data were not interpreted clinically since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.  相似文献   
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Glycoalkaloids were used as evidence of the affinities of nine taxa of Solanum Series Megistacrolobum and related potato cultigens from western Bolivia. S. boliviense, S. sanctae-rosae and S. toralapanum contain the commertetraose sugar moiety and appear to represent a relatively wild group within the Series. S. megistacrolobum, S. sogarandinum and S. raphanifolium show anomolous glycoalkaloid profiles that probably reflect hybridization associated with human disturbance. Primitive forms of the S. χ ajanhuiri cultigen are indistinguishable chemicaliy from conspecific weeds that were previously classified as S. megistacrolobum. Variation in total glycoalkaloid content within Series Megistacrolobum likely reflects direct selection by humans for reduced glycoalkaloid levels during the domestication process.  相似文献   
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