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Biomedical subjects

A Tsang

Publications and source records attributed to A Tsang.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1991

Biochemical and genetic characterization of a rapid-development strain in Dictyostelium discoideum.

We have examined the rates of development of six wild-type and rapid-development strains of Dictyostelium discoideum. Strains NC4 and HU1231, a derivative of V12, have very similar developmental profiles. In comparison to these two strains, amoebae of the rapid-development strain HT100 reach the aggregation stage about 6 h earlier. Also, intracellular cAMP levels in HT100 increase precociously during early development. Postaggregative morphogenesis of HT100 proceeds at the same rate as NC4 and HU1231. In addition we have assessed the progress of development by RNA hybridization blotting. The appearance in HT100 of mRNAs associated with aggregation is advanced by 6 h while the appearance of early gene products is not affected appreciably. These data suggest that HT100 differs from strains NC4 and HU1231 primarily in the process of aggregation. We have partially characterized the genetic background of strain HT100. Mating and cell fusion analyses suggest that HT100 is a derivative of V12. We constructed a growth-temperature-sensitive derivative of HT100, and fused it with HU1231 cells. The resulting diploids develop at the same rate as HT100, suggesting that the rapid-development phenotype in HT100 is dominant.

Animals↗
PubMed1990

Cloning and characterization of cDNAs encoding a novel cyclic AMP-binding protein in Dictyostelium discoideum.

The cellular slime mould, Dictyostelium discoideum, contains a novel cyclic AMP-binding protein, CABP1, which is composed of two subunits. Using anti-CABP1 monoclonal antibody as a probe, a cDNA clone was isolated from a lambda gt11 expression library. By hybrid selection of the complementary mRNA and its translation in vitro, we demonstrated that the cDNA hybridized to mRNAs encoding both CABP1 polypeptides. With the positive cDNA as a probe, we isolated a series of overlapping cDNA clones covering the coding region of both CABP1 mRNAs. Expression of the cloned cDNAs in bacteria and sequence analysis showed that the CABP1 subunits are identical in amino acid (aa) sequence, except that the small subunit is missing 37 aa near its N terminus. Genomic analysis suggested that the two CABP1 transcripts are derived from a single gene. The N-terminal half of each subunit is rich in proline, glutamine and glycine residues and contains a large block of aa repeats. The C-terminal half has an approx. 47% aa identity (86% with functionally conservative substitutions) with two polypeptides encoded by a plasmid determinant for tellurium anion resistance.

Amino Acid Sequence↗
PubMed1990

The molecular basis for alternative splicing of the CABP1 transcripts in Dictyostelium discoideum.

We have determined the nucleotide sequence of the CABP1 gene from Dictyostelium discoideum. Together with previous data on cDNA sequences, we establish that alternative splicing of transcripts derived from this gene is responsible for the production of the two CABP1 subunits. RNA blot analysis suggested that alternative splicing of the CABP1 transcripts occurs during growth and throughout development. In addition, we have compiled the intron sequences of Dictyostelium pre-mRNAs and observed that the GUAAGU hexanucleotide at the 5' splice site is highly conserved. The 5' splice site of CABP1 deviates from the consensus hexanucleotide in having a sequence of GUAAUA. To assess the role of the modified 5' splice on differential splicing, we have constructed an actin-CABP1 fusion gene and transformed it into Dictyostelium cells. Analysis by immunoprecipitation, with anti-CABP1 antibody and amplification of specific cDNAs by polymerase chain reaction show that the transcripts generated by the fusion gene are alternatively spliced. When the 5' splice site of the fusion gene is mutated to conform to the consensus sequence, the resulting transcripts are constitutively spliced. These observations suggest that changes in positions 5 and 6 of the donor splice site are involved in the alternative splicing of the CABP1 transcripts.

Actins↗
PubMed1990

Early controlled active mobilization with dynamic splintage for treatment of extensor tendon injuries.

Early controlled active mobilization with a dynamic splint for treatment of repaired extensor tendon injuries was studied prospectively. Postoperative dynamic splintage started on the third postoperative day and involved elastic band extension and active flexion of the digits. Mobilization of the digits within the splint was done immediately under supervision. Range of motion was stepped up progressively and splintage was stopped after 5 to 6 weeks. Thirty-eight patients with 48 digits were reviewed. The average follow-up was 6.7 months (range, 4 to 10 months). Injuries involving the fingers showed an average final total active motion of 229 degrees (range, 95 to 270 degrees), whereas injuries involving the thumb was 118 degrees (range, 78 to 150 degrees). Lesions distal to the knuckles (zones II, III, IV) showed the worst results with an average total active motion of only 188 degrees (range, 95 to 270 degrees). The duration out of work was 8.5 weeks (range, 4 to 25 weeks). There was no infection or acute tendon rupture. One patient required reconstruction of a persistent button-hole deformity; two required tenolysis and capsulotomy. Of the six unsatisfactory results with poor total active motion, four had injuries distal to the metacarpophalangeal joint, three of which were crushing injuries.

Adolescent↗
PubMed1988

Characterization of an unusual cAMP receptor and its related polypeptides in Dictyostelium discoideum.

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.

Dictyostelium↗
PubMed1988

Therapeutic functional bracing in upper limb fracture-dislocations.

The authors utilized the principle of functional bracing in the management of 307 fractures and/or dislocations of the upper limb. There were 29 humeral fractures, 111 fracture-dislocations around the elbow, 32 fractures of the forearm, and 135 fractures of the distal radius. The average follow-up was 9 months. The results showed that more than 95% of the patients regained good range of motion and power. The details of the technique and the protocols are described. The conclusion drawn from an analysis of the results is that functional bracing is a safe and effective method in the management of upper limb fracture dislocations.

Adolescent↗