Biomedical subjects
Publications and source records attributed to G R Taylor.
Enzymatic methods for mutation scanning still lack the sensitivity and specificity of the chemical cleavage of mismatch method. However developments in our understanding of the mismatch recognition process should lead to improvements. Several promising candidates exist with potential for more specific and sensitive mutation detection.
The present study was carried out to examine the effect of the daily intake of 10 g inulin on fasting blood lipid, glucose and insulin levels in healthy middle-aged men and women with moderately raised total plasma cholesterol (TC) and triacylglycerol (TAG) levels. This study was a double-blind randomized placebo-controlled parallel study in which fifty-four middle-aged subjects received either inulin or placebo for a period of 8 weeks. Fasting blood samples were collected before the supplementation period (baseline samples 1 and 2, separated by 1 week) and at weeks 4 and 8, with a follow-up at week 12. Compared with baseline values, insulin concentrations were significantly lower at 4 weeks (P < 0.01) in the inulin group. There was a trend for TAG values, compared with baseline, to be lower in the inulin group at 8 weeks (P < 0.08) returning to baseline concentrations at week 12. On comparison of the inulin and placebo groups, the fasting TAG responses over the 8-week test period were shown to be significantly different (P < 0.05, repeated measures ANOVA), which was largely due to lower plasma TAG levels in the inulin group at week 8. The percentage change in TAG levels in the inulin group during the 8-week study was shown to correlate with the initial TAG level of the subjects (rs -0.499, P = 0.004). We therefore conclude that the daily addition of 10 g inulin to the diet significantly reduced fasting insulin concentrations during the 8-week test period and resulted in lower plasma TAG levels, particularly in subjects in whom fasting TAG levels were greater than 1.5 mmol/l. These data support findings from animal studies that fructans influence the formation and/or degradation of TAG-rich lipoprotein particles, and the insulin data are also consistent with recent studies showing attenuation of insulin levels in fructan-treated rats.
The nuclear transcription factor-kappaB (NF-kappaB) and free radicals are known to be involved in apoptosis. We studied the effects of a series of di-aryl-substituted pyrazole NF-kappaB inhibitors including tepoxalin on tumor necrosis factor alpha (TNFalpha)-induced apoptosis in murine fibrosarcoma WEHI 164 cells. We found that potent inhibitors of NF-kappaB were also effective in attenuating apoptosis. WEHI 164 cells that had been dually treated with tepoxalin and the antioxidant pyrrolidine dithiocarbamate (PDTC) were significantly protected from TNFalpha-induced killing. To study the role of free radicals in mediating TNFalpha-induced apoptosis, stable WEHI 164 cells overexpressing Bcl-2, an antioxidant protein, were generated. These cells were protected from TNFalpha-induced apoptosis and neither tepoxalin nor PDTC provided further significant protection. These results suggest that Bcl-2, PDTC, and tepoxalin may attenuate apoptosis in this system by affecting the same signaling pathway or converging pathways. Because tepoxalin suppresses the release of free radicals, PDTC scavenges free radicals and Bcl-2 is an antioxidant protein, free radicals are among the key mediators of this TNF-induced killing event. Tepoxalin and antioxidants may be useful in developing new therapeutics for treating neurodegenerative diseases, autoimmune deficiency syndrome, and ischemia-reperfusion injuries.
After a decade of intensive use as an in vitro alternative to cloning DNA, PCR is now well established as the default method for DNA and RNA analysis. Recent developments have consolidated this position by the introduction of more robust formats, improvements in thermal cyclers and labelling and detection methods. The trend is towards increasing automation, although comparatively few diagnostic kits based on PCR are in wide use. At the same time the applications of PCR are being extended with modifications such as long, accurate PCR and arrayed oligonucleotides or expressed sequences.
Since the early work of Mann and Spoerry, probiotics in the form of fermented milk products have been reputed to have cholesterol-lowering properties in humans. However, studies conducted since the early 1970s have produced equivocal findings, with interpretation of the outcomes complicated by use of excessive quantities of product, inadequate sample sizes, failure to control nutrient intake and energy expenditure and variations in baseline blood lipids. More recent studies are of better quality, but fail to provide convincing evidence that 'live' fermented milk products have cholesterol-lowering efficacy in man. Future studies using probiotics should ensure adequate sample sizes sufficient to detect relatively small changes in blood cholesterol and should be conducted over longer periods of time. The recent introduction of the concept of prebiotics has directed attention towards the possibility that alterations in gut microflora induced by the fermentation of non-digestible components of the diet may also have the potential to influence systemic lipid metabolism. This possibility has been strengthened by the observation that in animals, dietary oligofructosaccharides cause suppression of hepatic triglyceride and VLDL synthesis, resulting in marked reductions in triglyceride, and to a lesser extent cholesterol, levels. Evidence for similar effects in humans is sparse and more studies are needed, particularly with respect to effects on postprandial triglyceride concentrations.
Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.
The methyl-directed long patch repair pathway in Escherichia coli is involved in increasing the fidelity of replication specific repair of DNA polymerase incorporation errors. This pathway is mediated by three gene products, MutS, MutL, and MutH, which are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). A MutS homologue has recently been identified in the extremely thermophilic bacterium, Thermus thermophilus. Here we describe analysis of the binding properties of this protein, which has indicated it can identify all specific base mismatches as well as one, two and three base pair insertion/deletion mutations. We therefore believe this protein may be generally useful for applications involving mismatch detection.
The human MSH-2 gene product is a member of a highly conserved family of proteins involved in post-replication mismatch repair. Germline mutations in this gene have been implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. We have previously shown that a region of high homology between MutS proteins of different species containing a nucleotide binding domain, is sufficient to bind DNA containing specific mismatched residues. In order to determine the minimal domain of hMSH-2 necessary for binding mismatch-containing oligonucleotides, deletion analysis of the C-terminal region was performed. We have constructed a 5' and 3' deletion series, expressed each deletion as a bacterial fusion protein and assessed it for ATPase activity and its ability to identify mismatch containing DNA. Here we demonstrate that a 585 bp fragment encoding 195 amino acids within the C-terminal domain of hMSH-2 is sufficient to bind to DNA containing mismatches.
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The results of immunological analyses before, during and after spaceflight, have established the fact that spaceflight can result in a blunting of the immune mechanisms of human crew members and animal test species. There is some evidence that the immune function changes in short-term flights resemble those occurring after acute stress, while the changes during long-term flights resemble those caused by chronic stress. In addition, this blunting of the immune function occurs concomitant with a relative increase in potentially infectious microorganisms in the space cabin environment. This combination of events results in an increased probability of inflight infectious events. The realization of this probability has been shown to be partially negated by the judicious use of a preflight health stabilization program and other operational countermeasures. The continuation of these countermeasures, as well as microbial and immunological monitoring, are recommended for continued spaceflight safety.
PURPOSE: To investigate donor cell survival following corneal limbal stem cell grafting, which is based on the corneal stem cell model. METHODS: We describe the use of the amelogenin gene probe with the polymerase chain reaction (PCR) to detect surviving donor cells and report preliminary studies using Y-specific DNA probes. RESULTS AND CONCLUSIONS: DNA polymorphisms have a detection limit of 10%. The SRY 'Y-specific' probe has a theoretical detection limit of 1 cell in 10,000. The techniques were applied to investigate survival of male donor cells in an aniridic female patient 2 1/2 years following limbal stem cell grafts. We speculate that low levels of donor-derived cells may still be present. We discuss the advantages and disadvantages of the two approaches, which may have future clinical and experimental application.
We report the six-year results of a prospective, controlled demographic trial of developmental dysplasia of the hip (DDH) treated in the Pavlik harness using ultrasound supervision. Our aim was to assess the value of ultrasound and its role in monitoring reduction in the harness, in terms of progression or failure of reduction at an early state. From 1988 to 1994, a total of 221 patients with 370 ultrasonographically abnormal hips was treated in the Pavlik harness. This represents a treatment rate for the Southampton district of 5.1 per 1000 live births. Sixteen hips in 12 patients were not reduced in the harness and required surgical treatment; 95.7% were successfully reduced. One case of mild avascular necrosis (0.3%) was identified in those treated by harness alone. Of the 221 patients 87.8% remain under radiological review, with 3.2% of affected hips showing continued, mild acetabular dysplasia. We conclude that ultrasound monitoring has led to an acceptably low level of intervention, a high reduction rate and minimal iatrogenic complications. The trial is continuing.
The genes involved in postreplicative DNA mismatch repair are a highly conserved family of proteins. In humans, germline mutations in these genes (hMSH-2, hMLH-1, hPMS-1, and hPMS-2) have been implicated in hereditary nonpolyposis colorectal cancer (HNPCC). We have previously shown that a region of high homology between the members of this class of proteins in different species contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues (1). To identify residues which are necessary for this activity, we have created a range of mutants containing amino acid substitutions within the nucleotide binding domain of hMSH-2. These mutants have been expressed and assessed for ATPase activity and their ability to identify mismatch-containing DNA. Here we demonstrate that a variant protein which has the conserved residue Lys 675 within the nucleotide binding consensus sequence altered to an alanine has severely impaired ATPase activity and is unable to bind DNA containing specific mismatched residues.
The human MSH-2 gene product is a member of a highly conserved family of proteins which are involved in post-replication mismatch repair. hMSH-2 is homologous to Escherichia coli (E. coli) MutS and Sacchromyces cerevisiae MSH-1 and MSH-2 proteins, which recognise heteroduplex DNA at the sites of all single base mismatches and deletions or insertions up to 4 base pairs. hMSH-2 is one of the hereditary non-polyposis colorectal cancer (HNPCC) tumor suppressor genes, and maps to human chromosome 2p16. Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. It has been shown that purified hMSH-2 binds specifically to nucleotide mismatches in double-stranded DNA. Here we demonstrate that a region of high homology between the members of this class of proteins contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues.
Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.
Bed rest, both with and without head-down tilt, has been extensively used as an earth-bound analog to study physiologic effects mimicking those occurring in weightlessness during spaceflight. We have been able to show in six subjects that 4 weeks of head-down tilt bed rest induces a significant decrease in interleukin-2 secretion by PHA-stimulated T lymphocytes. Another study, lasting 113 days, with two subjects showed a decreased interleukin-2 receptor expression in PHA-stimulated peripheral blood mononuclear cells but a decreased interleukin-2 production in one subject only. Under the same conditions, interleukin-1 production was largely increased in both subjects. Several other immune parameters were also analyzed. Increased interleukin-1 production could contribute to bone mineral loss encountered during bed rest and decreased interleukin-2 secretion could play a role in the appearance of infectious diseases often observed during bed red.
During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.
The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.