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Biomedical subjects

J M Scott

Publications and source records attributed to J M Scott.

At least 37 records · Page 2Linked to original sources
PubMed2000

The relationship between increased folate catabolism and the increased requirement for folate in pregnancy.

OBJECTIVES: To estimate the rate of folate catabolism in pregnant and nonpregnant women and to derive the recommended dietary allowance for folate. DESIGN: Prospective, observational study. SETTING: Rotunda Hospital, Dublin. WOMEN: Twenty-four healthy gravid women were studied once during each trimester and postpartum. Twenty-five nonpregnant controls were assessed before and after folic acid supplementation. INTERVENTIONS: Women provided 24-hour urine collections while adhering to a strict dietary regimen containing no exogenous folate catabolites. MAIN OUTCOME MEASURES: Urinary levels of p-acetamidobenzoylglutamate and p-aminobenzoylglutamate were measured by high pressure liquid chromatography. RESULTS: The 24-hour excretion of folate catabolites, expressed as mean [95% CI] folate equivalents in microg) progressively increased during pregnancy. A peak was reached in the third trimester (349.1 microg [308.1 to 390.1]) where the rate was more than twice the rate in the nonpregnant control group (136.4 microg [112.4 to 160.4]) (P < 0.001). Based on our results the recommended dietary allowance for folate in nonpregnant women should be 250 microg and this should rise during pregnancy to 430 microg in the second trimester and 540 microg in the third trimester. CONCLUSIONS: The rate of folate catabolism progressively increases during pregnancy reaching a peak in the third trimester at the time of maximal fetal growth. The increased demand for folate during pregnancy appears to be due to the accelerated breakdown of the vitamin because of its participation in cellular biosynthesis. These results provide a quantitative basis for the current debate on the appropriate recommended dietary allowance for folate in both pregnant and nonpregnant women.

4-Aminobenzoic Acid↗
PubMed2000

The Bacillus subtilis GTP binding protein obg and regulators of the sigma(B) stress response transcription factor cofractionate with ribosomes.

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of the sigma(B) transcription factor. We investigated Obg's cellular associations by differential centrifugation of crude B. subtilis extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a B. subtilis strain in which Obg was fused to green fluorescent protein. The results indicated that Obg is part of a large cytoplasmic complex. In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of B. subtilis lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay. Probing the gel filtration fractions with antibodies specific for sigma(B) and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the sigma(B) stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits. The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of sigma(B).

Bacillus subtilis↗
PubMed2000

Stress triggers a process that limits activation of the Bacillus subtilis stress transcription factor sigma(B).

Stress-induced activation of the Bacillus subtilis transcription factor sigma(B) is transitory. To determine whether the process that limits sigma(B) activation is itself triggered by stress, B. subtilis strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of sigma(B) activity. Without ethanol treatment, the induced cultures displayed continuously high sigma(B) activity. Ethanol treatment restricted ongoing sigma(B) activity, but only in strains with intact rsbX and -S genes. The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect. The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict sigma(B) induction following stress.

Adult↗
PubMed2000

Evaluation of novel assays in clinical chemistry: quantification of plasma total homocysteine.

BACKGROUND: There is a need for systematic evaluation of methods before their release to the market. We addressed this problem in novel homocysteine assays as part of an European Demonstration Project involving six centers in four countries. METHODS: Two immunological methods for measurement of plasma total homocysteine (P-tHcy), the fluorescence polarization immunoassay (FPIA) and the enzyme immunoassay (EIA), were compared with two comparison methods, HPLC and gas chromatography-mass spectrometry (GC-MS). All laboratories performed the following procedures: (a) familiarization; (b) determination of linearity and precision by analyzing five plasma samples with interrelated concentrations for 20 days; (c) correlation using patients' samples; and (d) assessment of long-term performance. RESULTS: Both immunological methods were linear for P-tHcy between 5 and 45 micromol/L. The intralaboratory imprecision (CV) was <5% for FPIA and <9% for EIA used with a sample processor. The bias was -2% to 3% for FPIA and 2-4% for EIA used with a sample processor. CONCLUSIONS: The immunological methods provide results with little bias compared with HPLC and GC-MS. The imprecision of the assays must be considered in the context of their intended use(s).

Fluorescence Polarization Immunoassay↗
PubMed1999

Methylenetetrahydrofolate reductase thermolabile variant and oral clefts.

Folic acid can prevent neural tube defects; in some cases the mechanism is probably a correction of a metabolic defect caused by thermolabile methylenetetrahydrofolate reductase (MTHFR) found in increased frequency in cases. It is less clear whether folic acid can prevent oral clefts, in part because it is not known whether thermolabile MTHFR is more common in those with oral clefts. This study examined the prevalence of the mutation (677 C-->T) that causes thermolabile MTHFR in subjects with oral clefts from a national Irish support group, and an anonymous control group randomly selected from a neonatal screening program covering all births in Ireland. Eighty-three of 848 control subjects were homozygous (TT) thermolabile MTHFR (9.8%). This defect was almost three times as common in the 27 subjects (25.9%) with isolated cleft palate (odds ratio 3.23, 95% confidence interval 1.32 -7.86, P = 0. 02) and somewhat more common in the 66 subjects with cleft lip with or without cleft palate (15.2%, odds ratio 1.65, 95% confidence interval 0.81-3.35, P = 0.20). When the two groups with different etiologies were combined, the overall odds ratio was 2.06 (95% confidence interval 1.16-3.66, P = 0.02). In the Irish population homozygosity for the common folate-related polymorphism associated with thermolabile MTHFR is significantly more frequent in those with isolated cleft palate, and could be etiologically important. Am. J. Med. Genet. 86:71-74, 1999. Published 1999 Wiley-Liss, Inc.

Child↗
PubMed1999

A Bacillus-specific factor is needed to trigger the stress-activated phosphatase/kinase cascade of sigmaB induction.

The general stress regulon of Bacillus subtilis is controlled by the transcription factor sigmaB. Environmental stress activates sigmaB via a phosphatase/kinase cascade that triggers sigmaB's release from an anti sigma factor complex. To determine if the members of the phosphatase/kinase cascade are sufficient to detect environmental stress and activate sigmaB, we expressed sigmaB and its regulators in E. coli. In E. coli, as in B. subtilis, the intact collection of regulators silenced sigmaB, while allowing sigmaB to be active if the cascade's most upstream negative regulator was deleted. The regulators could not, however, activate sigmaB in response to ethanol treatment or heat shock. In other experiments, the GroEL and DnaK chaperones, known to be important in controlling stress sigma factors in E. coli, were found to be unimportant for sigmaB activity in B. subtilis. The findings argue that stress induction of sigmaB requires novel factors that are B. subtilis specific.

ATP-Dependent Proteases↗
PubMed1999

Folate status and neural tube defects.

Periconceptional folic acid supplementation prevents approximately 70% of neural tube defects (NTDs). While most women carrying affected fetuses do not have deficient blood folate levels, the risk of having an NTD affected child is inversely correlated with pregnancy red cell folate levels. Current research is focused on the discovery of genetic abnormalities in folate related enzymes which might explain the role of folate in NTD prevention. The first candidate gene to emerge was the C677T variant of 5,10-methylenetetrahydrofolate reductase. Normal subjects who are homozygous for the mutation (TT) have red cell folate status some 20% lower than expected. It is now established that the prevalence of the TT genotype is significantly higher among spina bifida cases and their parents. Nevertheless, our studies show that the variant does not account for the reduced blood folate levels in many NTD affected mothers. We conclude that low maternal folate status may in itself be the most important risk factor for NTDs and that food fortification may be the only population strategy of benefit in the effort to eliminate NTDs.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1999

Methionine synthase: high-resolution mapping of the human gene and evaluation as a candidate locus for neural tube defects.

Periconceptual folate supplementation has been found to prevent the occurrence of many neural tube defects (NTDs). Consequently, genetic variation in folate metabolism genes is expected to contribute to the risk for neural tube defects. Methionine synthase catalyzes the vitamin B(12)-dependent conversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. The observation that homocysteine and vitamin B(12) levels are independent predictors of NTD risk suggested that methionine synthase could be a candidate gene for NTDs. To assess the role of the MS gene in NTDs, we performed high-resolution physical mapping of the MS locus, isolated highly polymorphic markers linked to the MS gene, and tested for an association between specific MS alleles and NTDs. We mapped the MS gene to a position between 909 and 913 cR(10000) on chromosome 1 by radiation hybrid mapping. Polymorphic markers D1S1567 and D1S1568 map to locations no more than 900 and 194 kb from the MS gene, respectively. The segregation of these polymorphic markers was measured in 85 Irish NTD families. No allele of either marker showed a significant association with NTDs using the transmission disequilibrium test. A lack of association was also observed for the D1919G missense mutation within the gene. Our results suggest that inherited variation in the MS gene does not contribute to NTD risk in this population.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Folate and vitamin B12.

The folates are made up of a pterdine ring attached to a p-aminobenzoate and a polyglutamyl chain. The active form is tetrahydrofolate which can have C1 units enzymically attached. These C1 units (as a formly group) are passed on to enzymes in the purine pathway that insert the C-2 and C-8 into the purine ring. A methylene group (-CH2-) attached to tetrahydrofolate is used to convert the uracil-type pyrimidine base found in RNA into the thymine base found in DNA. A further folate cofactor, i.e. 5-methyltetrahydrofolate, is involved in the remethylation of the homocysteine produced in the methylation cycle back to methionine. After activation to S-adenosylmethionine this acts as a methyl donor for the dozens of different methyltransferases present in all cells. Folate deficiency results in reduction of purine and pyrimidine biosynthesis and consequently DNA biosynthesis and cell division. This process is most easily seen in a reduction of erythrocytes causing anaemia. Reduction in the methylation cycle has multiple effects less easy to identify. One such effect is certainly on the nerve cells, because interruption of the methylation cycle causing neuropathy can also happen in vitamin B12 deficiency due to reduced activity of the vitamin B12-dependent enzyme methionine synthase (EC 2.1.1.13). In vitamin B12 deficiency, blocking of the methylation cycle causes the folate cofactors in the cell to become trapped as 5-methyltetrahydrofolate. This process in turn produces a pseudo folate deficiency in such cells, preventing cell division and giving rise to an anaemia identical to that seen in folate deficiency.

Diet↗
PubMed1999

The "thermolabile" variant of methylenetetrahydrofolate reductase and neural tube defects: An evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother.

Recent reports have implicated the "thermolabile" (T) variant of methylenetetrahydrofolate reductase (MTHFR) in the causation of folate-dependent neural tube defects (NTDs). We report herein the largest genetic study of NTD cases (n=271) and families (n=218) to date, establishing that, in Ireland, the "TT" genotype is found in 18.8% of cases versus 8.3% of controls (odds ratio 2.57; confidence interval [CI] 1.48-4.45; P=.0005). The maternal and paternal TT genotypes have intermediate frequencies of 13.8% and 11.9%, respectively, indicating that the predominant MTHFR-related genetic effect acts via the TT genotype of the developing embryo. Analysis of the 218 family triads of mother, father, and affected child with log-linear models supports this interpretation, providing significant evidence that the case TT genotype is associated with NTDs (P=.02) but no evidence of a maternal TT genotypic effect (P=. 83). The log-linear model predicted that the risk of NTDs conferred by the case TT genotype is 1.61 (CI 1.06-2.46), consistent with the paramount importance of the case TT genotype in determining risk. There is no compelling evidence for more than a modest additional risk conferred by a maternal TT genotype. These results favor a biological model of MTHFR-related NTD pathogenesis in which suboptimal maternal folate status imposes biochemical stress on the developing embryo, a stress it is ill-equipped to tolerate if it has a TT genotype.

Alleles↗
PubMed1999

Fortification with low amounts of folic acid makes a significant difference in folate status in young women: implications for the prevention of neural tube defects.

BACKGROUND: Mandatory fortification of grain products with folic acid was introduced recently in the United States, a policy expected to result in a mean additional intake of 100 microgram/d. One way of predicting the effectiveness of this measure is to determine the effect of removing a similar amount of folic acid as fortified food from the diets of young women who had been electively exposed to chronic fortification. OBJECTIVE: The objective was to examine the effect on folate status of foods fortified with low amounts of folic acid. DESIGN: We investigated the changes in dietary intakes and in red blood cell and serum concentrations of folate in response to removing folic acid-fortified foods for 12 wk from the diets of women who reportedly consumed such foods at least once weekly (consumers). RESULTS: Consumers (n = 21) had higher total folate intakes (P = 0.002) and red blood cell folate concentrations (P = 0.023) than nonconsumers (women who consumed folic acid-fortified foods less than once weekly; n = 30). Of greater interest, a 12-wk intervention involving the exclusion of these foods resulted in a decrease in folate intake of 78 +/- 56 microgram/d (P < 0.001), which was reflected in a significant reduction in red blood cell folate concentrations (P < 0.05). CONCLUSIONS: Cessation of eating folic acid-fortified foods resulted in removing 78 microgram folic acid/d from the diet. Over 12 wk this resulted in a lowering of red blood cell folate concentrations by 111 nmol/L (49 microgram/L). This magnitude of change in folate status in women can be anticipated as a result of the new US fortification legislation and is predicted to have a significant, although not optimal, effect in preventing neural tube defects.

Adolescent↗
PubMed1999

Antagonism of rapacuronium using edrophonium or neostigmine: pharmacodynamics and pharmacokinetics.

We have studied the pharmacodynamics and pharmacokinetics of rapacuronium (Org 9487) in 70 healthy patients. Neuromuscular transmission was monitored using TOF stimulation of the ulnar nerve and mechanomyography of the adductor pollicis muscle. Half of the patients were given a single dose of rapacuronium 1.5 mg kg-1 and the remainder rapacuronium 1.5 mg kg-1 with three incremental doses of 0.5 mg kg-1, each given when T1/T0 had recovered to 25%. In all patients, neuromuscular block was antagonized using neostigmine 0.05 mg kg-1 or edrophonium 1.0 mg kg-1 (allocated randomly), 2 min after the final dose of rapacuronium. All patients developed complete block after rapacuronium 1.5 mg kg-1. Mean onset time was 66 (SD 24) s. In patients who received an antagonist 2 min after the first dose of rapacuronium, time to recovery of T1/T0 to 25% was similar after neostigmine (9.8 (3.8) min) and edrophonium (10.3 (4.3) min): in patients who received incremental doses of rapacuronium, spontaneous recovery of T1/T0 to 25% after the first dose was 18.9 (4.7) min. In those who received an antagonist 2 min after the first dose of rapacuronium, times to recovery of T4/T1 to 0.7 were also similar after neostigmine (23.7 (7.7) min) and edrophonium (29.1 (10.7) min). After three incremental doses of rapacuronium, there was a longer time to recovery of T1/T0 = 25% after neostigmine compared with edrophonium (5.1 (1.0) vs 3.3 (1.3) min; P < 0.05) but more rapid recovery to T1/T0 = 75% (10.1 (2.9) vs 16.8 (10.1) min; P < 0.05) and T4/T1 = 0.7 (19.8 (6.3) vs 35.1 (10.4) min; P < 0.05). A three-compartment pharmacokinetic model was justified. Typical values for clearance and initial volume of distribution (V1) were 4.4 ml kg-1 min-1 and 94.8 ml kg-1, respectively. In females, clearance was decreased by 38.5% compared with males and V1 was decreased by 25% in patients aged more than 65 yr.

Adolescent↗
PubMed1999

The influence of 5,10 methylenetetrahydrofolate reductase genotypes on enzyme activity in placental tissue.

OBJECTIVE: To study the effects of heterozygosity and homozygosity for the C677T mutation of the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene on the activity of this enzyme in placental tissue from pregnancies not affected by neural tube defect. DESIGN: Placental tissue was genotyped for the C677T variants of MTHFR. Total enzyme activity and residual activity after heating to 46 degrees C for 5 minutes was then measured. SETTING: A teaching hospital. SAMPLE: Placental samples (n = 200), one from each of 200 uncomplicated term deliveries. MAIN OUTCOME MEASURES: Total and residual enzyme activity for MTHFR. RESULTS: Placentae heterozygous for the C677T mutation of the MTHFR gene had significantly lower total enzyme activity than those without the mutation; the lowest activity occurred in homozygotes for the mutation. The same pattern was seen in relation to enzyme activity after heating. CONCLUSION: This study demonstrates that reduced enzyme activity is associated with the C677T variant of MTHFR in placental tissue. This is an important metabolic step in folic acid metabolism and pro-

Biomarkers↗
PubMed1999

Methylation of cortical brain proteins from patients with HIV infection.

OBJECTIVES: Experimental models of vitamin B12 deficient-neuropathy are characterized by central nervous system protein hypomethylation. The encephalitis/vacuolar myelopathy complicating HIV infection and subacute combined degeneration of the cord due to vitamin B12 deficiency share similar biochemical and pathological abnormalities. Altered central nervous system methylation may be important in the pathogenesis of HIV encephalitis. To test this hypothesis we compared brain protein methylation of HIV-positive, and control, subjects. MATERIALS AND METHODS: Carboxymethyltransferase activity was assayed in postmortem cortical brain samples obtained from 16 control patients (9 males); mean age (59+/-5.1 years, range 21-87 years), 9 HIV-positive patients (7 males, 6 IVDA, 3 homosexual, 4 with HIV encephalitis, mean age 37, range 23-45), and 3 patients with Alzheimer's disease (mean age 78 years). RESULTS: The amount of radiolabelled SAM (S-adenosylmethionine) incorporated into carboxymethyl, and N-methylation sites within brain proteins from cortical white matter in vitro was significantly lower (P<0.05) in the HIV+ group vs controls. Carboxymethyltransferase activity was similar in the HIV-infected brains irrespective of the presence or absence of HIV encephalitis. Mean cortical methyl group incorporation was also lower in the Alzheimer's disease group compared to controls. CONCLUSION: The observation of reduced in vitro methylation of brain proteins from patients with HIV infection and Alzheimer's disease suggests that fewer unmethylated sites exist due to relative protein hypermethylation in vivo. The absence of hypomethylation in the brains of patients with HIV encephalitis suggests that hypomethylation is not necessary for the development of HIV encephalitis.

Adult↗
PubMed1999

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX). B. subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway. Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.

Adenosine Triphosphate↗
PubMed1999

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity.

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.

ATP-Dependent Proteases↗
PubMed1999

Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease, and its clinical implications.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with an increased incidence of thromboembolic disease. Hyperhomocysteinaemia (hyper-tHcy), a condition associated with the C677T variant of 5, 10-methylenetetrahydrofolate reductase (MTHFR), is linked with an increased incidence of thromboembolic disease. Hyper-tHcy has been reported in patients with IBD. AIMS: To assess the prevalence of the C677T MTHFR genotype and the contribution of this genotype to hyper-tHcy in patients with IBD. METHODS: Patients with established IBD (n=174) and healthy controls (n=273) were studied. DNA samples were genotyped for the MTHFR (C677T) mutation. Subjects were categorised as homozygous for the thermolabile variant (TT), heterozygous for wild type and variant (CT), or homozygous for the wild type (CC). RESULTS: Plasma homocysteine concentrations were significantly higher in patients with IBD than in healthy controls. A total of 17.5% of ulcerative colitis and 16.8% of Crohn's disease patients were homozygous for the C677T variant compared with 7.3% of controls. Homozygosity (TT) for the variant was associated with higher plasma tHcy levels in patients with IBD and in healthy controls. When all subjects who were TT for the variant were excluded, median plasma tHcy was still significantly higher in IBD than controls. Plasma vitamin B(12) levels were lower in patients with IBD irrespective of MTHFR genotype. CONCLUSIONS: There is an association between the thermolabile MTHFR C677T variant and IBD. This accounts in part for the raised plasma tHcy found in patients with IBD and may contribute to the increased incidence of thromboembolic complications. All patients with IBD should receive low dose folic acid and vitamin B(12) therapy to protect against the thromboembolic complications of raised tHcy.

Adolescent↗