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Biomedical subjects

R L Davies

Publications and source records attributed to R L Davies.

At least 37 records · Page 2Linked to original sources
PubMed1991

Optimal conditions for the analysis of Pasteurella haemolytica lipopolysaccharide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The optimal conditions for the analysis of the lipopolysaccharide (LPS) of two serotype A1 isolates and a serotype A2 isolate of Pasteurella haemolytica by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were determined. The LPS of the A1 isolates possessed O side chains, consisting of high molecular mass bands with the appearance of a ladder-like pattern, as well as a low molecular mass core-oligosaccharide region; the LPS of the A2 isolate consisted only of the core-oligosaccharide region. Furthermore, the LPS of the two A1 isolates differed in the core-oligosaccharide region. Optimal resolution of low molecular mass LPS components was obtained in a 15% acrylamide resolving gel containing 4 M urea whereas optimal resolution of high molecular mass components was obtained when urea was omitted. Conventional silver staining resulted in excellent visualisation of LPS bands, whereas a modified staining method did not detect additional bands, as has been demonstrated with the LPS of Pseudomonas aeruginosa. Proteinase K digestion of outer membranes gave more clearly defined LPS profiles than did similar digestions of whole cells, and more closely resembled the profiles of purified LPS. With the exception of slight variation in the average molecular mass of a group of O side chains between logarithmic and stationary phases there were no differences in LPS profiles at various stages of the growth cycle; freezing and thawing of LPS samples had no effect on the profiles.

Animals↗
PubMed1991

Effects of regional myocardial ischaemia on coronary artery calibre in the rabbit.

Microangiographic and electron microscopic studies were undertaken in isolated, perfused rabbit hearts to examine the effects of acute regional myocardial ischaemia on coronary artery calibre, vascular resistance and ultrastructure. Selective left coronary angiograms were recorded prior to, after 90 min of acute regional myocardial ischaemia induced by occlusion of the marginal left ventricular branch of the circumflex artery, and after reperfusion for 5 min. Vasodilation occurred in the proximal left anterior descending artery (9 +/- 1%, P less than 0.01) and the unoccluded part of the marginal branch (21 +/- 1%, P less than 0.001) during ischaemia which reversed on reperfusion. During reperfusion, coronary vascular resistance increased by 43 +/- 10% (P less than 0.001) despite mild vasodilation of vessels in the ischaemic zone (up to 10 +/- 1%, P less than 0.01). Failure of reperfusion occurred in vessels smaller than 30 microns in diameter (terminal arterioles) and was associated with ultrastructural damage in the ischaemic zone which was particularly prominent at the apex. These findings suggest that failure of reperfusion following 90 min of acute regional myocardial ischaemia in the rabbit is associated with damage to terminal arterioles and capillaries and that the calibre of the larger coronary vessels is maintained. Failure of reperfusion after prolonged myocardial ischaemia may occur despite arterial dilatation in the ischaemic zone.

Animals↗
PubMed1991

Outer membrane protein profiles of Yersinia ruckeri.

The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.

Animals↗
PubMed1991

Virulence and serum-resistance in different clonal groups and serotypes of Yersinia ruckeri.

The virulence in rainbow trout (Oncorhynchus mykiss) of 32 isolates of Yersinia ruckeri, representing a range of biotypes, serotypes, and OMP-types, was examined. Virulence was assayed in fish of average weight 7.7 g by bath challenge for 1 h with approximately 5 x 10(7) cells per ml. Two of the six serotype O1 clonal groups of Y. ruckeri, clones 2 and 5, were virulent, whereas the other four clonal groups, clones 1, 3, 4 and 6, as well as all serotype O2, O5, O6 and O7 isolates examined, were avirulent. Analysis of susceptibility to the bactericidal effect of non-immune rainbow trout serum demonstrated an association between virulence and serum resistance. The virulent serotype O1 clonal groups were serum resistant, whereas the avirulent serotype O1 clonal groups and other serotypes were, with some exceptions, serum sensitive. The fact that some serum resistant isolates were avirulent suggested that other factors may be required for the full expression of virulence. The study also demonstrated that rainbow trout and brook trout (Salvelinus fontinalis) differ in their susceptibility to Y. ruckeri.

Animals↗
PubMed1991

The protective role of eicosapentaenoic acid [EPA] in the pathogenesis of nephrolithiasis.

The low incidence of atherosclerosis and other degenerative diseases including stone disease in the Greenland Eskimo has been attributed to their high consumption of oily fish with its high concentration of eicosapentaenoic acid (EPA). Man cannot synthesis EPA from the precursor essential fatty acid, linolenic acid, and can only assimilate preformed EPA present in fish and fish oil, to bring about a change in the pathway of eicosanoid metabolism from the n-6 to the n-3 series. With a westernised diet the oxygenated products of renal prostaglandin synthesis are metabolites of the n-6 series and these are known to play an important role in several pathophysiological states including stone disease. Our previous studies have shown a relationship between prostaglandin activity and urinary calcium excretion and it would seem that the initiating factor/s for stone formation trigger the mechanisms for prostaglandin synthesis resulting in the biochemical abnormalities associated with stone disease. The Eskimo may be protected from these events by possession of an eicosanoid metabolism that follows an n-3 pathway. To test this hypothesis experiments were performed using an animal model of nephrocalcinosis. The animals were divided into three groups; one group was given an intra-peritoneal injection of 10% calcium gluconate daily for 10 days to induce nephrocalcinosis; a second group was fed MaxEPA fish oil before and during the calcium gluconate injections and a third group only received an intra-peritoneal injection of N saline. A group of 12 recurrent, hypercalciuric/hyperoxaluric stone-formers were treated with fish oil for eight weeks to study the effects on solute excretion. Nephrocalcinosis, which was readily produced in the control animals, was prevented in the experimental animals by pre-treatment with fish oil and urine calcium excretion was significantly reduced. The urinary calcium and oxalate excretion in the recurrent, hypercalciuric stone-formers was significantly reduced with fish oil treatment over an eight week period. There were no untoward side-effects. These studies indicate that the incorporation of EPA in the diet as a substitute metabolic pathway could be a unique way of correcting the biochemical abnormalities of idiopathic urolithiasis.

Adult↗
PubMed1990

Comparison of methods for the analysis of outer membrane antigens of Neisseria meningitidis by western blotting.

The method of extraction of outer membrane proteins (OMPs), the conditions of electrophoretic transfer, and the conditions of antibody binding, were compared in Western blotting studies of Neisseria meningitidis outer membrane antigens. The OMP profiles obtained by SDS-PAGE of outer membrane vesicles extracted with lithium chloride/sodium acetate were compared with profiles obtained by Sarkosyl extraction; these profiles were further compared with the patterns obtained by 125I-labelling of surface-exposed proteins. Sarkosyl extracts gave profiles most closely resembling those of 125I-labelled whole-cells and gave the best resolution of the major proteins. After transfer in Tris-glycine-methanol buffer some proteins, including the major proteins, were not completely transferred and remained in the gel, with the class 2/3 and 5 proteins not effectively detected on nitrocellulose by amido black staining. There was weak antibody recognition of the class 1 and 4 proteins but good recognition of lipooligosaccharide (LOS) and H8 antigen. Empigen BB had no effect on renaturation of the class 1 protein. When 0.1% SDS was incorporated in the same buffer all of the proteins were removed from the gel, and although the major proteins bound to nitrocellulose other proteins did not. There was weak antibody recognition of the class 1 and 4 proteins, stronger reaction to the class 5 protein, but no recognition of the class 2 protein, LOS or H8 antigen, Empigen BB slightly enhanced antibody recognition of the class 1 protein. After transfer in Tris-glycine buffer, all the major proteins were transferred and bound to nitrocellulose and, other than the class 2 protein, were recognised by antibody, both in the presence or absence of Empigen BB, as were LOS and the H8 antigen. Differences existed in the patterns of antibody recognition between the lithium and the Sarkosyl extracts; additional proteins were recognised in the lithium extracts. The surface-labelling studies indicated, however, that some of these proteins were not surface-exposed. Some minor proteins appeared to be more highly immunogenic than the major proteins.

Acetates↗
PubMed1990

O-serotyping of Yersinia ruckeri with special emphasis on European isolates.

Yersinia ruckeri is the aetiological agent of enteric redmouth disease (ERM), an acute to chronic bacteraemic infection of salmonid fish. The O-serotypes of 127 isolates of Y. ruckeri obtained from Europe (96 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), as well as four reference strains, were determined by slide agglutination test and microplate agglutination assay. A serotyping scheme is proposed based on heat-stable O-antigens; the serotypes were designated O1, O2, O5, O6 and O7. The proposed scheme is compared to serotyping schemes described by other authors. All five O-serotypes were present in both Europe and North America, whereas only serotype O1 isolates were identified in Australia and South Africa. These findings suggest that European and North American populations of Y. ruckeri are interrelated, thus supporting previous evidence which suggests that the organism was introduced into Europe from North America by the importation of asymptomatic infected carrier fish. Conversely, the results suggest that Australia and South Africa are more isolated from the dissemination of Y. ruckeri. Serotypes O5, O6 and O7 have not previously been recognized in Europe and these findings will have important implications on the diagnosis of ERM and on the vaccination of fish against this disease. It is suggested that the Australian isolate previously described as serotype III is a rough-type mutant and that other isolates described in the literature as serotype III have been incorrectly serotyped and are, in fact, serotype O1. To avoid further confusion it is suggested that the scheme described here be adopted for serological studies of Y. ruckeri.

Agglutination Tests↗
PubMed1990

Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1.

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.

Aflatoxin B1↗
PubMed1990

Origin and structure of benign prostatic hyperplasia.

The development of microradiography of the prostate gland has provided a new approach to investigating the origin and structure of benign hyperplasia. It has demonstrated that benign prostatic hyperplasia is usually microadenomatous in structure, although reticular types do occur. It originates at the lateral boundary of the periurethral muscle in the position of the transitional zone. In contrast, stromal nodules are homogeneous and amorphous in structure and develop at the site of the short straight urethral ducts within the periurethral muscle.

Adolescent↗
PubMed1989

The role of EDRF in flow distribution: a microangiographic study of the rabbit isolated ear.

A microangiographic technique was used to study the influence of endothelium-derived relaxing factor (EDRF) on vasomotor control mechanisms in resistance vessels of intact buffer-perfused rabbit ear. Selective inhibition of EDRF activity by hemoglobin unmasked an intrinsic ("myogenic") constrictor response to sudden increases in flow rate. EDRF activity was greatest in arteries in which calculated shear stress and hydraulic resistance were maximal, namely the central ear artery and its first generation of branch arteries: these are proximal "feed" vessels (150-700 microns internal diameter) in this bed. The findings are consistent with enhancement of EDRF release by the physical stimulus of shear stress in resistance vessels as previously demonstrated in conduit vessels-a phenomenon which is likely to exert a major influence on flow in vascular networks. EDRF activity thus reduced perfusion pressure and power losses, particularly in highly constricted preparations. Shear-induced release of EDRF may provide an integrating link between flow and arterial topography by optimizing perfusion characteristics over a wide range of flow rates.

Angiography↗
PubMed1989

Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays.

We have demonstrated that the human cytochrome P1-450 gene can be transfected into the AHH-1 human lymphoblastoid cell line using the pHEBo vector and hygromycin selection. The transfected gene was expressed when regulatory sequences derived from the herpes simplex virus thymidine kinase gene were incorporated in appropriate orientations. Gene expression was monitored at the enzyme level using assays for 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase and benzo[a]pyrene hydroxylase activities. Bulk transformed cell populations had 2- to 3-fold more of these enzyme activities compared with control populations. Subclones of the bulk population expressing still higher levels of 7-ethoxyresorufin deethylase activity were also obtained. Expression of the transfected cytochrome P1-450 gene was stable for 20-30 days in the presence of hygromycin B. The transformed cell populations were found to be suitable for use in gene locus mutation assays and the mutagenicity of aflatoxin-B1 and 2-acetylaminofluorene (AAF) were examined. Aflatoxin-B1 was found to be 2-3 times more mutagenic to cells bearing the transfected cytochrome P1-450 activity as compared with control cells. In contrast, no difference in AAF mutagenicity was observed. Analysis of the AAF metabolite profile indicated that cells expressing the transfected cytochrome P1-450 gene produced 8-fold more N- and 7-hydroxy-AAF than control cells. The similarity in mutagenic responses between control cells and cells bearing the transfected cytochrome P1-450 gene may be due to the low deacetylase activity of AHH-1 cells. These observations indicate that this vector and expression system are suitable for introducing novel metabolic activities into the AHH-1 cell line.

Cell Line↗
PubMed1989

Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens.

We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.

Benzo(a)pyrene↗
PubMed1989

Structure and per-rectal ultrasonography of prostatic disease using cadaver specimens.

One hundred cadaver prostates were examined by per-rectal ultrasound and the findings correlated with the microradiographic and histopathological features of whole sections taken at the levels of scanning. The normal gland consists of both periurethral muscle and glands. The periurethral muscle has little structure on microradiography and is therefore hypoechoic (low amplitude echoes) on ultrasound scanning. The gland itself, both central and peripheral zones, has a lattice appearance on microradiography (reticular structure) and therefore produces mid-range amplitude echoes on ultrasound. Benign hypertrophy develops at the edge of the periurethral muscle and is microadenomatous in structure. This produces mid-range amplitude echoes which are coarser than in the normal gland. Associated calcification has a characteristic distribution around the adenomas. Cancer produces an amorphous structure resulting in loss of ultrasonic echoes (hypoechoic). In a few cancers irregular calcification is present, resulting in high echoes in the ultrasound scan (hyperechoic) which are not in the typical benign distribution. The internal structure of the prostate was therefore demonstrated in its histopathological states by the technique of microradiography and seen to relate to its representation by ultrasound.

Adolescent↗
PubMed1989

Comparative histopathology, microradiography and per-rectal ultrasonography of the prostate using cadaver specimens.

A technique for examining cadaver prostates by per-rectal ultrasonography has been developed. The ultrasonic plane was defined and corresponding sections taken for microradiographic and histopathological examination. The technique has enabled a comparison to be made of the structural changes that occur in the prostate in disease states and the corresponding ultrasonic features.

Adolescent↗
PubMed1989

Adonis microcarpa (pheasant's eye) toxicity in pigs fed field pea screenings.

Seed of Adonis microcarpa (pheasant's eye) fed at 5.6 g/kg of the diet induced virtually total feed refusal within 3 d in growing and finishing pigs. It also caused vomiting, rapid and shallow breathing and death in a minority. These effects were probably caused by cardiac glycosides whose structure and effects closely resemble those of digoxin. Feed intake and growth recovered within 2 weeks of removal of the seed.

Animal Feed↗
PubMed1989

Microangiographic investigation of the effects of radiographic contrast media and hyperkalaemia on coronary artery calibre in the rabbit.

A microangiographic technique, using novel methods of contact X-ray microscopy, was developed and used to study the effects of ionic (Urografin 370) and non-ionic (Omnipaque 350) radiographic contrast media, and hyperkalaemia (exposure to 15, 30 and 60 mM-K+ solutions) on coronary artery calibre and total coronary vascular resistance in isolated, Langendorff-perfused rabbit hearts. Repeated injection of both types of contrast media produced similar haemodynamic effects and some reduction in coronary diameter, but greater variation in arterial calibre was observed with Urografin (P less than 0.001). Perfusion with hyperkalaemic solutions produced a dose-related increase in total coronary vascular resistance (P less than 0.001), but this was associated with a complex pattern of changes in large-vessel calibre. The direction and magnitude of the responses to hyperkalaemia varied depending on the site within the artery studied; in general hyperkalaemia produced a dose-related vasoconstriction which was greater distally than proximally. Thus, with 15 mM-K+ vasoconstriction occurred in the left anterior descending artery (4.3% proximally; 15.6% distally) which increased to 40.7 and 52.1% (proximally and distally, respectively) with 30 mM-K+. With 60 mM-K+ further diffuse and segmental vasoconstriction occurred in 4/5 and 1/5 hearts respectively (mean values 31.5 and 56.7% at the proximal and distal sites). During perfusion with 15 mM-K+, net vasodilatation (17% at both the proximal and distal sites) was noted in the left ventricular branch of the circumflex artery, but with 30 mM-K+ vasoconstriction (18.3% proximally and 38.0% distally) occurred. Thus, while hyperkalaemia consistently increased coronary vascular resistance, these changes were associated with marked variation in the extent and distribution of in situ constriction and dilatation within the coronary circulation.

Angiography↗