Biomedical subjects

Alan P Lewis

Publications and source records attributed to Alan P Lewis.

5 recordsLinked to original sources

PubMed2005

DING proteins are from Pseudomonas.

DING proteins have been described as animal and plant proteins with potential biomineralisation, receptor or signalling roles that have been characterised by an N-terminal DINGGG-sequence. However, these sequences have only ever been identified as either N-terminal peptides or partial cDNA sequences, and have yet to be detected in any of the many genomic animal and plant genomes now available. Microbial relatives of the DING proteins have been described, which appear to be periplasmic phosphate-binding proteins. Recently, full-length Pseudomonas aeruginosa UCBPP-PA14 and Hypericum perforatum genes have been sequenced that show high homology to the published DING protein N-terminal sequences, and small peptides previously identified in conjunction with the peptide sequencing of DING proteins can also be mapped to regions across these full-length sequences. Searching with these sequences identifies other plant and animal cDNA fragments in the public nucleotide databases, and, additionally, an unordered rat genomic contig that contains a DING-like sequence on a small fragment. Analysing the codon usage of these DNA sequences identifies all of these sequences as of Pseudomonas origin, suggesting that DING proteins do not exist in eukaryotes, but instead are potentially due to microbial contamination or infection.

Amino Acid Sequence↗

PubMed2005

hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore are not ion channels.

Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right.

Animals↗

PubMed2004

Differential gene expression identifies novel markers of CD4+ and CD8+ T cell activation following stimulation by Mycobacterium tuberculosis.

T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor's CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.

CD4-Positive T-Lymphocytes↗

PubMed2002

Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence.

A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.

Animals↗

PubMed2000

Comparison of the proteome of Mycobacterium tuberculosis strain H37Rv with clinical isolate CDC 1551.

The genome sequences of two virulent strains of Mycobacterium tuberculosis (H37Rv and CDC 1551) are now available. CDC 1551 is a recent clinical isolate and H37Rv is a commonly used lab strain which has been subject to in vitro passage. The two strains have been shown to display differing phenotypes both in vivo and in vitro. The proteome of the two strains grown in liquid culture were examined over time to determine whether there are any major differences between them at the protein level and the differences were compared to the genome data. Total cell lysates of the two strains were analysed by two-dimensional electrophoresis. Approximately 1750 protein spots were visualized by silver staining and the protein profiles of the two strains were found to be highly similar. Out of a total of 17 protein spot differences, seven were unique to CDC 1551 and three to H37Rv. Two further spots showed increased intensity in H37Rv, one spot showed differing vertical mobility between the strains and four showed differing spot intensities with time. Twelve of the spot differences were identified using mass spectrometry; however, no obvious association with phenotype could be deduced. When genome differences were analysed and related to the proteome differences, a mobility shift identified in the MoxR protein could be explained by a point mutation at the gene level. This proteome analysis reveals that, despite having been maintained under vastly different conditions, namely in vitro passage and in vivo transmission, these two strains have remained highly similar.

Bacterial Proteins↗