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31.
GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.  相似文献   
32.
OBJECTIVE--To compare the prevalence of antibody to and proviral DNA of the retrovirus HTLV-I in relatives of 11 British patients with tropical spastic paraparesis who had migrated from Jamaica before they developed symptoms, and to examine factors possibly related to transmission of HTLV-I. DESIGN--Migrant, family study. Antibody state was determined by several methods and confirmed by western blotting; the polymerase chain reaction was used to detect proviral DNA. SETTING--Britain and Jamaica. SUBJECTS--All available first degree relatives: those born and still resident in Jamaica (group 1); those born in Jamaica who migrated to Britain (group 2); and index patients'' children who were born and resident in Britain (group 3). All had been breast fed and none had had blood transfusions. RESULTS--Of the 66 living relatives, 60 were traced. Seroprevalence among those born in Jamaica (irrespective of current residence) was 22% (10/46; 95% confidence limits 9 to 34%) compared with zero among British born offspring (0/14) and was higher in group 2 at 33% (7/21; 12 to 55%) than in group 1 at 12% (3/25; 0 to 25%). (Patients in group 1 had the greatest mean age.) Proviral DNA was not detected in any subject negative for HTLV-I antibody, making prolonged viral incubation in those negative for the antibody unlikely. CONCLUSION--In this sample factors related to place of birth and early residence were more important in transmission of HTLV-I than maternal or age effects. In areas with a low to moderate prevalence policies of preventing mothers who are carriers of the virus from breast feeding would be premature.  相似文献   
33.
Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.  相似文献   
34.
The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.  相似文献   
35.
The developmental expression of adrenocortical steroid hydroxylases was studied in bovine fetuses from 40 to 280 days gestational age. The expression of P-450(17 alpha) is first detected at a gestational age of 50 days and reaches a maximum at 60-70 days. The expression of P-450(17 alpha) then declines and is nondetectable at a gestational age of 100 days. P-450(17 alpha) is not expressed again until about 240 days, i.e. shortly before birth (approximately 280 days). P-450scc, P-450c21, P-450(11 beta) and adrenodoxin were present in fetal adrenals throughout gestation. This "on-off-on" pattern of P-450(17 alpha) expression during fetal development was associated with a corresponding episodic production of cortisol. Immunoreactive corticotropin (ACTH) levels in fetal plasma were elevated in small fetuses (corresponding to less than or equal to 100 days) and in near-term fetuses (corresponding to greater than 250 days) compared with those in mid-gestation fetuses. In primary culture, adrenal cells from mid-gestation fetuses contained no detectable P-450(17 alpha) but rapidly responded to ACTH with an increase in P-450(17 alpha) protein and mRNA. The tissue specificity of the developmental patterns is emphasized by the fact that both P-450(17 alpha) and P-450scc were detectable throughout the development of the fetal testes, whereas only P-450scc was detectable in fetal bovine ovary prior to 200 days. Thus, in fetal bovine adrenal it appears that ACTH is the major regulatory factor effecting the intermittent presence of P-450(17 alpha), whereas the presence of the other steroid hydroxylases is either regulated by additional factors or shows a much different sensitivity to ACTH.  相似文献   
36.
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent Km values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (Ki = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.  相似文献   
37.
BBC microcomputer controlled field inversion gel electrophoresis   总被引:1,自引:0,他引:1  
Agarose gel electrophoresis to separate DNA molecules is a widelyused technique in molecular biology but there is an upper limitto the sizes that can be resolved. Pulsed field techniques haveextended this limit but require expensive equipment. Here wedescribe a home-made control unit to interface conventionalelectrophoresis equipment to a BBC microcomputer for the purposesof field inversion gel electrophoresis. Received on October 6, 1987; accepted on November 10, 1987  相似文献   
38.
The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.  相似文献   
39.
Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.  相似文献   
40.
Human neonates were exposed to an artificial odorant for 22h within the first two days after birth. When tested on days16–18 postpartum, these infants displayed preferentialorientation to the exposure odor when paired with a novel odorant.The effects of early mere exposure on the stimulus propertiesof odors can therefore endure over a two-week interval, indicatingthat infants retain a memory trace of the exposure odor throughoutthat time period.  相似文献   
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