Biomedical subjects
Publications and source records attributed to David Coates.
Hip replacement surgery is becoming increasingly specialised and evidence shows that, particularly in revision, surgery is best done by specialists. An audit of GPs suggests great variation in referral practice, with one-third prepared to refer to a 'generalist' orthopaedic surgeon. GPs need better communication with hospitals and knowledge of where specialists are available.
Angiotensin converting enzyme 1, found widely throughout the animal kingdom, is an integral membrane bound protein whose active sites are directed to the extracellular spaces. Two isoforms are expressed in mammals, a single domain germinal isoform required for male fertility, and a double domain somatic isoform which has a key role in the renin-angiotensin system. Both somatic domains are active with different substrate affinities. Mouse knockout experiments, and comparative work with invertebrate homologues, suggest that the two domains have clearly distinct roles. The importance of therapies involving inhibition of angiotensin converting enzyme are undisputed, but our understanding of how and why these therapies work is now being informed by the tools of genomic and comparative biology.
The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis naïve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.
Protease data mining can take advantage both of the many specialist, Web-available databases that cover the genetic, protein and nucleic acid sequence information that is specific to a variety of organisms, and of a flexible, but defined, classification system. However, precomputed data, such as gene predictions, should be used with care. Unless there is definitive supporting information, ideally sequencing of a cDNA to show that the predictions are accurate, followed by expression and biochemical characterization of the predicted protein, the predicted gene and its product remains a possibility, rather than a certainty.
The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.