Search PubMed⌕ Search

Biomedical subjects

C M Thomas

Publications and source records attributed to C M Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1999

Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB.

KorA and KorB proteins of IncP1 plasmid RK2 are encoded in the central control region (ccr) of the plasmid and act as global regulators of plasmid genes for replication, transfer and stable inheritance. KorA represses seven promoters on RK2, by binding to a defined operator site, OA, which always occurs in promoter regions. KorB recognises another operator, OB, which is found 12 times on the RK2 genome, but not always in promoter regions. At five of the KorA-regulated promoters, an OBsequence is also present. The presence of both KorA and KorB leads to severely decreased promoter activity. By measuring repression at different levels of KorA and KorB alone and in combination, we showed that there is at least 3. 4-fold co-operativity between them at korApin vivo. Testing the ability of previously isolated KorA mutants to act in a co-operative way in the presence of KorB in vivo or in vitro showed that the C-terminal part of KorA between amino acid positions 68 and 83 is required for this co-operativity. This region is part of a segment that is highly conserved between KorA and two other RK2 proteins, TrbA and KlcB. We propose that this conserved region may provide the basis for co-operativity with KorB either indirectly, by modulating DNA structure near the KorB binding site, or directly by serving as the "recognition" patch of each protein by KorB. It may thus serve as a key domain in allowing a sensitive response of the global circuits to changes in repressor concentration and thus modulation of replication, transfer and maintenance.

Amino Acid Sequence↗
PubMed1999

Nonsyndromic orofacial clefts: association with maternal hyperhomocysteinemia.

Maternal folic acid supplementation has been suggested to play a role in the prevention of nonsyndromic orofacial clefts, i.e., cleft lip +/- cleft palate. Using a case-control design, we investigated vitamin-dependent homocysteine metabolism in 35 mothers with nonsyndromic orofacial cleft offspring and 56 control mothers with nonmalformed offspring. A standardized oral methionine loading test was performed, in which fasting and afterload plasma total homocysteine, serum and red-cell folate, serum vitamin B12, and whole-blood vitamin B6 levels were determined. We found that both fasting (P < 0.01) as well as afterload (P < 0.05) homocysteine concentrations were significantly higher in cases compared to controls. Hyperhomocysteinemia, defined by a fasting and/or afterload homocysteine concentration above the 97.5th percentile, was present in 15.6% of the cases and in 3.6% of controls (odds ratio, 5.3 (1.1-24.2)). The median concentrations of serum (P < 0. 01) and red-cell (P < 0.05) folate were significantly higher, and vitamin B6 concentrations appeared to be significantly lower (P < 0. 05), in cases compared with controls. No significant difference was observed between groups for vitamin B12. These preliminary data offer evidence that maternal hyperhomocysteinemia may be a risk factor for having nonsyndromic orofacial cleft offspring.

Adult↗
PubMed1999

Complete sequence of Bacillus subtilis plasmid p1414 and comparison with seven other plasmid types found in Russian soil isolates of Bacillus subtilis.

We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow. We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site. One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR). The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified. The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response. We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B. subtilis isolates. All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids. This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress. This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment.

Amino Acid Sequence↗
PubMed1999

An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography.

Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/1) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

Adult↗
PubMed1999

Low-dose folic acid supplementation does not influence plasma methionine concentrations in young non-pregnant women.

An elevated plasma total homocysteine (tHcy) concentration is a risk factor for cardiovascular disease and for having offspring with a neural-tube defect. Folate is a methyl donor in the remethylation of homocysteine into methionine. Although folic acid supplementation decreases tHcy concentrations, effects of folic acid supplementation on plasma methionine concentrations are unclear. There is also concern that folic acid supplementation negatively affects vitamin B12 status. We studied effects of low-dose folic acid supplementation on methionine and vitamin B12 concentrations in plasma. We also investigated whether baseline plasma methionine and tHcy concentrations correlated with the baseline folate and vitamin B12 status. For a period of 4 weeks, 144 young women received either 500 micrograms folic acid each day, or 500 micrograms folic acid and placebo tablets on alternate days, or a placebo tablet each day. Plasma methionine, tHcy and plasma vitamin B12 concentrations were measured at start and end of the intervention period. Folic acid supplementation had no effect on plasma methionine or plasma vitamin B12 concentrations although it significantly decreased tHcy concentrations. Plasma methionine concentrations showed no correlation with either tHcy concentrations (Spearman rs-0.01, P = 0.89), or any of the blood vitamin variables at baseline. Baseline tHcy concentrations showed a slight inverse correlation with baseline concentrations of plasma vitamin B12 (rs-0.25, P < 0.001), plasma folate (rs-0.24, P < 0.01) and erythrocyte folate (rs-0.19, P < 0.05). In conclusion, low-dose folic acid supplementation did not influence plasma methionine or plasma vitamin B12 concentrations. Furthermore, no correlation between plasma methionine concentrations and the blood folate and vitamin B12 status was shown.

Adolescent↗
PubMed1999

Repression at a distance by the global regulator KorB of promiscuous IncP plasmids.

KorB protein (358 amino acids) binds to 12 highly conserved sequences on the RK2 genome and co-ordinates the expression of at least five operons encoding genes for stable inheritance and plasmid transfer. KorB represses the trfA, korA and klaA promoters where it binds 4 bp upstream of the -35 region (class I KorB operators, OB). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where OB is between 80 and 189 bp away from the transcription start point (class II operator). A C-terminal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His6-tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing OB sequences. At the trbBp region, where OB9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp -35 region had no effect on the ability of KorB to repress trbBp. Nevertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of OB9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA polymerase may interact directly with KorB bound at OB9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.

Bacterial Proteins↗
PubMed1999

Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus.

The incC and korB genes of IncP-1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immunofluorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP-1 plasmids. When maintained by the low-copy-number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an efficient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP-1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one- and three-quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern fits with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning.

Animals↗
PubMed1999

Low-dose folic acid supplementation decreases plasma homocysteine concentrations: a randomized trial.

BACKGROUND: An elevated plasma total homocysteine concentration is a risk factor for cardiovascular disease and neural tube defects. A high daily intake of supplemental folic acid is known to decrease total homocysteine concentrations. OBJECTIVE: We studied the effect of low-dose folic acid administration (250 or 500 (microgram/d) for 4 wk on plasma total homocysteine concentrations and folate status. We also investigated whether total homocysteine concentrations and blood folate concentrations returned to baseline after an 8-wk washout period. DESIGN: In this placebo-controlled study, 144 healthy women aged 18-40 y received 500 microgram folic acid/d, 500 microgram folic acid every second day (250 microgram/d), or a placebo tablet with their habitual diet (mean dietary folate intake: 280 microgram/d). RESULTS: Administration of 250 and 500 microgram folic acid/d for 4 wk significantly increased folate concentrations in plasma (P < 0.001) and red blood cells (P < 0.01). Total homocysteine concentrations decreased significantly (P < 0.001) in women (n = 50) who took 250 microgram folic acid/d [mean (+/-SEM) deviation from baseline: - 11.4 +/- 198%] and in women (n = 45) who took 500 microgram folic acid/d (-21.8 + 1.49%). Eight weeks after the end of the intervention period (week 12), plasma total homocysteine concentrations in the folic acid-supplemented groups had not returned to baseline (week 0). CONCLUSIONS: Doses of folic acid as low as 250 microgram/d, on average, in addition to usual dietary intakes of folate significantly decreased plasma total homocysteine concentrations in healthy, young women. An 8-wk washout period was not sufficient for blood folate and plasma total homocysteine concentrations to return to baseline concentrations.

Adult↗
PubMed1999

Risk of persistent cobalamin deficiency in adolescents fed a macrobiotic diet in early life.

BACKGROUND: Cobalamin deficiency has been described in children consuming macrobiotic diets. OBJECTIVE: We investigated whether moderate consumption of animal products is sufficient for achieving normal cobalamin function in 73 adolescents who had received a macrobiotic diet until 6 y of age and had then switched to a lactovegetarian, lactoovovegetarian, or omnivorous diet (macrobiotic adolescents). DESIGN: Hematologic indexes and serum concentrations of methylmalonic acid (MMA), total homocysteine (tHcy), and folate were measured. Current consumption frequency of animal products and cobalamin intake from dairy products were assessed by questionnaire. Data from 94 age-matched adolescents who received an omnivorous diet from birth were used as a reference. RESULTS: Serum cobalamin concentrations were significantly lower and concentrations of MMA and folate and mean corpuscular volume (MCV) were significantly higher in macrobiotic adolescents than in control adolescents: of macrobiotic adolescents, 21% had abnormal MMA concentrations (>0.41 micromol/L), 37% had abnormal cobalamin concentrations (<218 pmol/L), 10% had abnormal tHcy concentrations (> 12.8 micromol/L), and 15% had abnormal MCV (> 89 fL). In macrobiotic adolescents, dairy products (200 g milk or yogurt and 22 g cheese/d) supplied on average 0.95 microg cobalamin/d; additionally, these adolescents consumed fish, meat, or chicken 2-3 times/wk. In girls, meat consumption contributed more to cobalamin status than the consumption of dairy products, whereas in boys these food groups were equally important. CONCLUSIONS: A substantial number of the formerly strict macrobiotic adolescents still had impaired cobalamin function. Thus, moderate consumption of animal products is not sufficient for restoring normal cobalamin status in subjects with inadequate cobalamin intake during the early years of life.

Adolescent↗
PubMed1999

Dietary folate from vegetables and citrus fruit decreases plasma homocysteine concentrations in humans in a dietary controlled trial.

Elevated total plasma homocysteine (tHcy) concentrations are considered a risk factor for neural tube defects (NTD) and cardiovascular disease. Supplementation with folic acid decreases the risk of women having children with NTD. In both sexes, it decreases tHcy levels. We investigated the efficacy of natural dietary folate in improving folate and homocysteine status. We performed a 4-wk dietary controlled, parallel design intervention trial with 66 healthy subjects (18-45 y) divided into 3 treatment groups: the dietary folate group, the folic acid group and the placebo group. Each day each group was fed a different diet. The dietary folate group received a diet high in vegetables and citrus fruit (total folate content approximately 560 microgram) plus a placebo tablet. The folic acid group received a diet naturally low in folate (approximately 210 microgram) plus 500 microgram folic acid and placebo tablet on alternate days, i.e., 250 microgram folic acid/d. And the placebo group received the same low-folate diet as the folic acid group plus a placebo tablet. After 4 wk of intervention, folate status improved, and tHcy concentrations decreased in both the dietary folate and the folic acid groups. From the amount of additional folate (350 microgram/d) and folic acid (250 microgram/d) consumed, the relative bioavailability of dietary folate compared to folic acid was calculated to be 60-98%, depending on the endpoint used. In conclusion, increasing the consumption of vegetables and citrus fruit, both good sources of folate, will improve folate status and decrease tHcy concentrations. This may contribute to the prevention of cardiovascular disease and NTD in the general population

Adolescent↗
PubMed1999

Homologues of the Cf-9 disease resistance gene (Hcr9s) are present at multiple loci on the short arm of tomato chromosome 1.

The tomato Cf-4 and Cf-9 genes map at a genetically complex locus on the short arm of chromosome 1 and confer resistance against Cladosporium fulvum through recognition of different pathogen-encoded avirulence determinants. Cf-4 and Cf-9 are members of a large gene family (Hcr9s, Homologues of Cladosporium fulvum resistance gene Cf-9), some of which encode additional distinct recognition specificities. A genetic analysis of the majority of Hcr9s suggests that their distribution is spatially restricted to the short arm of chromosome 1. Two loci of clustered Hcr9 genes have been analyzed physically that mapped distal (Northern Lights) and proximal (Southern Cross) to the Cf-4/9 locus (Milky Way). Sequence homologies between intergenic regions at Southern Cross and Milky Way indicate local Hcr9 duplication preceded cluster multiplication. The multiplication of clusters involved DNA flanking Hcr9 sequences as indicated by conserved lipoxygenase sequences at Southern Cross and Milky Way. The similar spatial distribution of Hcr9 clusters in different Lycopersicon spp. suggests Hcr9 cluster multiplication preceded speciation.

Base Sequence↗
PubMed1999

IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity In vivo and operator binding in vitro.

The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC and korB, belong to the parA and parB families, respectively, of genome partitioning functions. Both korB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of incC alone caused rapid displacement of RK2. Using two different reporter systems, we show that incC modulates the action of KorB. Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription by korB. This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with incC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

Bacterial Proteins↗
PubMed1999

Absence of prolactin gene expression in colorectal cancer.

AIMS: Previous studies documenting hyperprolactinaemia in patients with colorectal cancer have suggested that the tumour is the source of hormone production. The aim of this study was to determine the frequency of hyperprolactinaemia in patients with colorectal cancer before, during, and after surgery, and also to determine whether prolactin is produced by these tumours. METHODS: Serum prolactin concentrations were measured in 20 patients with colorectal cancer before, during, and after surgical resection of their tumours. Samples taken during surgery included peripheral venous blood and blood taken from the main veins draining the tumour. To determine whether the tumour was responsible for the production of prolactin in these patients, paraffin wax embedded sections of tumour specimens were subjected to immunohistochemistry and western blotting using a monoclonal antibody to prolactin. RESULTS: Five patients (three women, two men) had preoperative prolactin concentrations above the normal reference range, although this increase was of clinical importance in only two. After surgical resection of their tumours, prolactin concentrations remained high in both patients. All 20 patients had greatly raised prolactin values at the time of surgery, irrespective of whether this was measured in peripheral blood or in blood taken from veins draining the tumour. All 20 colorectal cancer tissue samples, including those with raised preoperative and/or postoperative prolactin concentrations, were negative for prolactin staining. Frozen tissue was also available in four cases. The absence of prolactin gene expression in these four tumours was confirmed both by repeat immunohistochemistry and by western blotting. A further 50 colorectal cancer cases examined by immunohistochemistry alone were also unreactive for prolactin. CONCLUSIONS: The results of this study suggest that serum prolactin concentrations may occasionally be raised in colorectal cancer patients, but that the tumour is not the source of hormone production.

Blotting, Western↗
PubMed1999

Aerobic training and cutaneous vasodilation in young and older men.

To determine the effect and underlying mechanisms of exercise training and the influence of age on the skin blood flow (SkBF) response to exercise in a hot environment, 22 young (Y; 18-30 yr) and 21 older (O; 61-78 yr) men were assigned to 16 wk of aerobic (A; YA, n = 8; OA, n = 11), resistance (R; YR, n = 7; OR, n = 3), or no training (C; YC, n = 7; OC, n = 7). Before and after treatment, subjects exercised at 60% of maximum oxygen consumption (VO2 max) on a cycle ergometer for 60 min at 36 degrees C. Cutaneous vascular conductance, defined as SkBF divided by mean arterial pressure, was monitored at control (vasoconstriction intact) and bretylium-treated (vasoconstriction blocked) sites on the forearm using laser-Doppler flowmetry. Forearm vascular conductance was calculated as forearm blood flow (venous occlusion plethysmography) divided by mean arterial pressure. Esophageal and skin temperatures were recorded. Only aerobic training (functionally defined a priori as a 5% or greater increase in VO2 max) produced a decrease in the mean body temperature threshold for increasing forearm vascular conductance (36.89 +/- 0.08 to 36.63 +/- 0.08 degrees C, P < 0.003) and cutaneous vascular conductance (36.91 +/- 0.08 to 36.65 +/- 0.08 degrees C, P < 0.004). Similar thresholds between control and bretylium-treated sites indicated that the decrease was mediated through the active vasodilator system. This shift was more pronounced in the older men who presented greater training-induced increases in VO2 max than did the young men (22 and 9%, respectively). In summary, older men improved their SkBF response to exercise-heat stress through the effect of aerobic training on the cutaneous vasodilator system.

Adolescent↗
PubMed1999

Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila.

The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known (beta)-spectrin isoforms: a conventional (beta)-spectrin chain, and the novel isoform (beta)(Heavy)-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two (beta)-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing (beta)(Heavy)-spectrin into which the basolateral (beta)-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of (beta)-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of (beta)(Heavy)-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms.

Animals↗