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Biomedical subjects

K M Donald

Publications and source records attributed to K M Donald.

8 recordsLinked to original sources
PubMed2003

Quantification of gene transcription and enzyme activity for functionally important proteolytic enzymes during early development in the Pacific oyster Crassostrea gigas.

Gene transcripts and enzyme activities were quantified for a selection of functionally important aminopeptidases at 2-day intervals throughout the first 72 days of development in the Pacific oyster Crassostrea gigas. Leucine aminopeptidase (LAP) and cathepsin B (CathB) gene transcripts were quantified using fluorogenic ('real time') PCR. LAP and CathB gene transcripts were detected at all time points. The proportion of CathB transcripts remained essentially constant and low throughout development (Ct<35). The proportion of LAP transcripts was often similar (Ct<30), but with a distinct peak in transcript abundance at day 19 (Ct approximately 23). CathB and cathepsin D (CathD) enzyme activities were measured biochemically. Whilst CathD activity peaked at day 19, LAP and CathB activities both peaked at day 24. The closely coupled increase in transcript and enzyme activity for LAP indicates regulation at the transcriptional level. Alternatively, the peak in enzyme activity for CathB without enhanced transcriptional activity suggests post-transcriptional regulation. Similar mechanisms of regulation for LAP and CathB have been observed in both plants and mammals, indicating widespread conservation.

Animals↗
PubMed2001

HIV gp120 receptors on human dendritic cells.

Dendritic cells (DCs) are important targets for human immunodeficiency virus (HIV) because of their roles during transmission and also maintenance of immune competence. Furthermore, DCs are a key cell in the development of HIV vaccines. In both these settings the mechanism of binding of the HIV envelope protein gp120 to DCs is of importance. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte-derived DCs (MDDCs) rather than CD4. In this study a novel biotinylated gp120 assay was used to determine whether CLR or CD4 were predominant receptors on MDDCs and ex vivo blood DCs. CLR bound more than 80% of gp120 on MDDCs, with residual binding attributable to CD4, reconfirming that CLRs were the major receptors for gp120 on MDDCs. However, in contrast to recent reports, gp120 binding to at least 3 CLRs was observed: DC-SIGN, mannose receptor, and unidentified trypsin resistant CLR(s). In marked contrast, freshly isolated and cultured CD11c(+ve) and CD11c(-ve) blood DCs only bound gp120 via CD4. In view of these marked differences between MDDCs and blood DCs, HIV capture by DCs and transfer mechanisms to T cells as well as potential antigenic processing pathways will need to be determined for each DC phenotype.

AIDS Vaccines↗
PubMed2001

Transcript analysis of the genes encoding aminopeptidase N and alanine aminotransferase, two enzymes involved in protein turnover, in the Pacific oyster, Crassostrea gigas.

Molecular probes have been developed to detect aminopeptidase N (ApN) and alanine aminotransferase (ALAT) transcripts in the Pacific oyster Crassostrea gigas. Degenerate primers were designed using ApN and ALAT sequences stored in the EMBL database. Amplification of C. gigas genomic DNA using these primers resulted in amplification of a 344-bp ApN fragment and a 530-bp alanine aminotransferase fragment. The deduced amino acid sequence of the ApN fragment displayed 75 and 73% identities with sequences of ApN from human and mouse, respectively. The deduced amino acid sequence of the ALAT fragment displayed 57% identity both with human and rat ALAT. An ApN transcript of approximately 3.1 kb was detected by northern blotting in larvae and in adult digestive gland and gonadal tissue. No transcript was detected in adult adductor muscle. An ALAT transcript of approximately 2 kb was similarly detected in larvae and in adult gonadal tissue, but was undetectable in adult digestive gland and adductor muscle. Transcript detection employing RT-PCR demonstrated low-level expression of both ApN and ALAT in all studied tissues, in both larvae and adults.

Alanine Transaminase↗
PubMed1997

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.

Carrier Proteins↗