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141.
Isolation and characterization of a mucin-glycoprotein from rat submandibular glands 总被引:3,自引:0,他引:3
L A Tabak L Mirels L D Monte A L Ridall M J Levine R E Loomis F Lindauer M S Reddy B J Baum 《Archives of biochemistry and biophysics》1985,242(2):383-392
A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present. 相似文献
142.
Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases 总被引:1,自引:0,他引:1
Eukaryotes and archaea both possess multiple genes coding for family B DNA
polymerases. In animals and fungi, three family B DNA polymerases, alpha,
delta, and epsilon, are responsible for replication of nuclear DNA. We used
a PCR-based approach to amplify and sequence phylogenetically conserved
regions of these three DNA polymerases from Giardia intestinalis and
Trichomonas vaginalis, representatives of early-diverging eukaryotic
lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs
suggests that the gene duplications that gave rise to the three replicative
paralogs occurred before the divergence of the earliest eukaryotic
lineages, and that all eukaryotes are likely to possess these paralogs. One
eukaryotic paralog, epsilon, consistently branches within archaeal
sequences to the exclusion of other eukaryotic paralogs, suggesting that an
epsilon-like family B DNA polymerase was ancestral to both archaea and
eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form
monophyletic groups in phylogenetic analysis, it is possible that archaeal
family B paralogs themselves evolved by a series of gene duplications
independent of the gene duplications that gave rise to eukaryotic paralogs.
相似文献
143.
Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration. 相似文献
144.
A heat shock response has been observed in a wide variety of eukaryotic organisms and may be universal. In Drosophila four heat shock proteins (hsp 22, 23, 26, and 27) have been found in nuclei (A. Arrigo, S. Fakan, and A. Tissieres, 1980, Develop. Biol. 78, 86–103). Eight heat shock-induced proteins of Dictyostelium discoideum were found to be preferentially localized in nuclei. They ranged in size from 26,000 to 32,000 daltons and could be recognized among the chromatin-associated proteins. Partial degradation of the chromatin released the low-molecular-weight heat shock proteins to the same extent as the histones. The heat shock response has been shown to result in protection of cells to the lethal effects of high temperature in a variety of organisms including Dictyostelium. We found that this response is extremely rapid in Dictyostelium being maximal by 30 min. The low-molecular-weight heat shock proteins enter the nuclei rapidly and so could play a role there in thermal protection. A mutant strain was isolated which is impaired in the protection afforded by a heat shock. This strain synthesizes most proteins normally but specifically fails to synthesize the low-molecular-weight heat shock proteins under conditions which result in their induction in wild-type cells. 相似文献
145.
146.
Rita R. Colwell Jafrul A.K. Hasan Anwarul Huq Lawrence Loomis Ronald J. Siebeling Miguel Torres Sylvia Galvez Sirajul Islam David Bernstein 《FEMS microbiology letters》1992,97(3):215-219
Cholera epidemics caused by Vibrio cholerae 01 continue to represent a major public health concern in many developing countries. A rapid and simple test kit for the detection of V. cholerae 01 has been developed. The kit, CholeraScreen is a monoclonal antibody-based, co-agglutination test and is used directly with stool specimens. It does not include culturing the specimen and is performed without the need for sophisticated laboratory equipment. Specificity of the test was demonstrated, using 118 reference cultures, to which cross-reactions were not observed. Preliminary results of field trials carried out in Guatemala and Bangladesh demonstrated that the test is equally sensitive as conventional culture methods in detecting V. cholerae and, in many cases, more sensitive. The CholeraScreen test is simple, specific, and does not require culturing procedures, making it suitable for direct detection of cells of V. cholerae in clinical specimens, even in the field. Also, the test requires less than five minutes to complete. 相似文献
147.
P Basta M B Strickland W Holmes C R Loomis L M Ballas D J Burns 《Biochimica et biophysica acta》1992,1132(2):154-160
Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies. 相似文献
148.
J J Baldassare P A Henderson D Burns C Loomis G J Fisher 《The Journal of biological chemistry》1992,267(22):15585-15590
Human platelets were found by immunoblot analysis to express protein kinase C (PKC) isozymes alpha, beta, delta, and zeta, but not gamma, epsilon, or eta. Exposure of platelets to thrombin, in the presence of 1 mM calcium, induced increased membrane association of PKC-alpha, -beta, and -zeta, while the subcellular distribution of PKC-delta remained unaltered. Maximal membrane association (2-fold) of PKC-alpha, -beta, and -zeta occurred within 1 min and was sustained for at least 10 min after the addition of thrombin. Similar results were obtained in the presence of the RGDS peptide, which blocks thrombin-induced binding of fibrinogen to its receptor, which indicates that PKC translocation was independent of fibrinogen binding. In the absence of added extracellular calcium, thrombin-induced translocation of PKC-alpha, -beta, and -zeta was transient, reaching a maximum at 1 min and returning to base line by 10 min. In the presence of calcium, thrombin induced a rapid (within 15 s) 8-fold rise in inositol 1,4,5-trisphosphate, which returned to baseline levels within 1 min, and a biphasic increase in sn-1,2-diacylglycerol (DAG), with peaks at 15 s and 2 min, which remained elevated for at least 5 min. Chelation of external calcium abolished the second phase of DAG formation but had no effect on the kinetics or magnitude of the increase in inositol 1,4,5-trisphosphate or the first phase of DAG formation. Two early PKC-dependent functions, serotonin release and 40-kDa protein phosphorylation, were independent of extracellular calcium and sustained DAG. These data demonstrate that in thrombin-stimulated human platelets the duration of the increased PKC membrane association closely parallels that of increased DAG content, and sustained elevations in DAG content and PKC translocation are dependent on extracellular calcium. 相似文献
149.
Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins. 相似文献
150.
Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine 总被引:30,自引:0,他引:30
A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs. 相似文献