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A Tsang

Publications and source records attributed to A Tsang.

At least 37 records · Page 2Linked to original sources
PubMed1996

A prespore-specific gene of Dictyostelium discoideum encodes the small subunit of ribonucleotide reductase.

We have isolated the gene. rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that beta-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.

Amino Acid Sequence↗
PubMed1996

Isolation by genetic complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger.

We have constructed an Aspergillus niger cDNA library with a yeast expression vector. The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4). Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis. Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B. Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes. Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase. In minimal medium, only leu2A expression was greatly stimulated. We examined the codon preference of these two genes. Whereas leu2A shows a bias in codon usage typical of A. niger genes, leu2B does not. These results indicate the presence in A. niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase.

3-Isopropylmalate Dehydrogenase↗
PubMed1994

Expression and characterization of a Dictyostelium discoideum annexin.

The annexins are calcium-dependent phospholipid-binding proteins. Recently the gene encoding the homologue of a mammalian annexin has been identified in Dictyostelium discoideum. Analysis of cDNA and genomic clones showed that the transcript for Dictyostelium annexin is alternatively spliced (Greenwood, M. and Tsang, A. (1991) Biochim. Biophys. Acta 1088, 429-432; Döring, V., Schleicher, M and Noegel, A. (1991) J. Biol. Chem. 266, 17509-17515). Here, we showed that the Dictyostelium annexin DNA hybridized to two populations of transcripts. We used a recombinant annexin polypeptide to raise polyclonal antibody. Immunoblot analysis revealed that the antibody recognized two polypeptides of 48 kDa and 54 kDa in developing D. discoideum cells. The molecular sizes of these polypeptides correspond well with the expected sizes of the alternatively spliced products. The 48-kDa and 54-kDa polypeptides were purified by isoelectric focusing to more than 70% homogeneity. The partially purified proteins were found to associate with phosphatidylserine vesicles in a calcium-dependent manner. These results suggest that the 48- and 54-kDa polypeptides are the products of alternative splicing of the annexin transcripts. During development the two polypeptides accumulate at different rates to about 60 times the level detected in vegetative cells. On the other hand, RNA blot analysis showed that the level of the annexin transcripts in multicellular aggregates was about 5 times that of vegetative cells.

Alternative Splicing↗
PubMed1993

A diabetic rabbit model for pig islet xenotransplantation.

An alloxan-diabetic rabbit model was established for the testing of the function of discordant xenogeneic pig islets isolated and purified from adult pig pancreata. The functional state of the pig islet transplants and immunological state of the rabbit recipients were assessed. Intraportal transplantation of 0.47 +/- 0.01 ml of pig islets with estimated 57418 +/- 5020 in number containing an estimated insulin content of 33.93 +/- 2.97 units (n = 7; mean +/- SEM) resulted in the normalization of blood glucose with a corresponding rise in insulin levels in the diabetic rabbit recipients for 2 days. An intravenous glucose tolerance test performed in 4 recipients during the normoglycemic period resulted in an improved K rate (2.5 +/- 0.4) over the diabetic controls, but this was significantly lower than the normal control animals (K rate = 4.5 +/- 0.4; n = 8). In vitro studies demonstrated that the preformed antibodies detected in the rabbit recipients were cytotoxic to the pig islet cells and lymphocytes. Heat treatment at 56 degrees C and mercaptoethanol treatment markedly reduced the cytotoxic activities of the sera. These findings implicated involvement of complement and IgM class antibodies in the killing of the pig islet cells. Furthermore, pig islet transplants at the kidney capsule site were coated with IgM class antibodies. This study has demonstrated that pig islets can be successfully isolated and purified in sufficient numbers for xenotransplantation studies in alloxan-diabetic rabbit. The porcine islet-to-alloxan diabetic rabbit combination can serve as a highly stringent and useful discordant model for assessing the effectiveness of various immunomodulation and immunosuppressive regimens. The finding of an optimal approach to immunorejection would potentially be applicable to actual clinical islet xenotransplantation in diabetic patients.

Animals↗
PubMed1993

Serovar determination and molecular taxonomic correlation in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum: a cooperative study of the International Working Group on Mycobacterial Taxonomy.

A cooperative study was conducted by the International Working Group on Mycobacterial Taxonomy to correlate the agglutination serovar designations of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains with the species ascriptions of these organisms according to molecular criteria and cultural properties and to assess the reproducibility of serovar determinations for a set of 63 reference strains of these species. Among the molecular criteria, the level of agreement between results obtained with nucleic acid probes and T-catalase serology results was 94% for strains of M. avium and M. intracellulare. Nucleic acid probes were not available for M. scrofulaceum, but none of the 10 strains ascribed to this species on the basis of catalase serology data reacted with a nucleic acid probe for M. avium or M. intracellulare. Ascription to a species on the basis of mycolic acid high-performance liquid chromatography patterns was in agreement with catalase serology results in 86% of the cases examined. Most strains belonging to serovars 1 through 6 and 8 through 11 were identified by molecular criteria as M. avium, most strains belonging to serovars 7, 12 through 20, 23, and 25 were identified as M. intracellulare, and most strains belonging to serovars 41 through 43 were identified as M. scrofulaceum, in agreement with common current practice. Evidence for assigning serovar 27 to M. scrofulaceum was obtained. However, two strains of a given serovar may, on occasion, be placed in different species. The dominant species assignments for strains belonging to serovars 21, 24, 26, and 28 remain unresolved.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination Tests↗
PubMed1992

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCl containing 0.1 mM DTT and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration-dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.

Animals↗
PubMed1992

Studies of a tumor-associated antigen, COX-1, recognized by a monoclonal antibody.

Monoclonal antibodies against an ovarian tumor cell line, OC-3-VGH, were generated using modified hybridoma technology. Among the seven that were selected for their high specificity and affinity to ovarian cancer cells and low cross-reactivity to most normal human tissues, RP 215 was shown to react specifically with a tumor-associated antigen, COX-1, from certain ovarian/cervical cancer cell lines. By Western blot assay, COX-1 was shown to have a subunit molecular mass of about 60 kDa and exist as an aggregate in the native state. COX-1 could also be detected in the shed medium of certain cultured tumor cells. A solid-phase sandwich enzyme-immunoassay procedure was designed for quantitative determinations of COX-1 in the shed medium or in patients' sera using RP 215 for both well-coating and the signal detection. Highly purified COX-1 was obtained from the shed medium of cultured OC-3-VGH tumor cells mainly by hydroxyapatite and immunoaffinity chromatography with RP 215 as the affinity ligand. At neutral pH, purified COX-1 also exists as an aggregate and is relatively stable at temperatures below 50 degrees C. Its immunoactivity was found to decrease with time in the presence of trypsin. However, the immunoactivity of COX-1 was not affected upon incubation with carbohydrate-digestive enzymes or concanavalin A and only partially inactivated in the presence of NaIO4 or iodoacetamide. Treatments of COX-1 with dithiothreitol and guanidine thiocyanate resulted in a complete loss of activity. Furthermore, rabbit antisera raised against purified COX-1 exhibited similar immunospecificity to that of RP 215. The results of this study suggest that COX-1 is a glycoprotein consisting of a 60 kDa subunit, which is recognized by RP 215 through its peptide determinant. Preliminary retrospective clinical studies were performed to assess the utility of a COX-1 enzyme immunoassay kit for detection and monitoring of patients with ovarian and cervical cancers.

Antibodies, Monoclonal↗
PubMed1991

Sequence and expression of annexin VII of Dictyostelium discoideum.

Sequence analysis reveals that a gene expressed during growth and early development of Dictyostelium discoideum encodes a polypeptide which exhibits extensive similarity with annexins, a family of calcium/phospholipid binding proteins. Comparison of the amino acid composition of the N-termini suggests that the Dictyostelium annexin is a homologue of human synexin, also referred to as annexin VII.

Amino Acid Sequence↗
PubMed1991

A maize gene expressed during embryogenesis is abscisic acid-inducible and highly conserved.

We have isolated by differential hybridization a cDNA, termed Emb564, which is complementary to an 800 nt embryo-specific transcript in Zea mays. The expression of Emb564 can be enhanced by exogenous abscisic acid (ABA) and the effect of ABA on the accumulation of Emb564 transcript appears to be confined to embryos at the early and mid embryonic stages. In addition, Emb564 is expressed at low levels in ABA-deficient but not in ABA non-responsive embryos. Genomic analysis suggested that the Emb564 mRNA is encoded by a single gene. Sequence analysis showed that Emb564 exhibits extensive similarities with several known ABA-inducible genes.

Abscisic Acid↗
PubMed1991

Disruption of the gene encoding the p34/31 polypeptides affects growth and development of Dictyostelium discoideum.

We have used homologous recombination to disrupt the gene which codes for p34 and p31, two polypeptides related to a cAMP-binding protein (CABP1) in Dictyostelium discoideum. By screening a total of 80 independent transformants by Southern blotting, four mutants have been isolated. Two of these mutants were analyzed in detail. Our results indicate that, while a null allele has not been obtained, both mutants express drastically reduced levels of truncated p34 and p31. Phenotypic analysis has demonstrated that both of them grow significantly more slowly than wild-type controls when bacteria are used as a food source. Interestingly, this growth defect is not seen when the cells are cultured axenically. In addition, the mutants possess an altered developmental profile. They complete development approximately 3 h later than wild-type controls. These results indicate that p34 and p31 play roles in both growth and development in this organism.

Alleles↗
PubMed1991

Isolation and characterization of cDNA clones encoding polypeptides related to a Dictyostelium discoideum cyclic AMP binding protein.

By screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.

Actins↗