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J M Scott

Publications and source records attributed to J M Scott.

At least 73 records · Page 4Linked to original sources
PubMed1997

Are common mutations of cystathionine beta-synthase involved in the aetiology of neural tube defects?

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defect (NTD) pregnancies. Plasma levels of Hcy are governed by both genetic and nutritional factors and the aetiology of NTDs is also known to have both genetic and nutritional components. We therefore examined the frequency of relatively common mutations in the enzyme cystathionine beta-synthase (CBS), which is one of the main enzymes that controls Hcy levels, in the NTD population. Neither the severely dysfunctional G307S CBS allele nor the recently reported 68 bp insertion/I278T CBS allele was observed at increased frequency in the cases relative to controls. We therefore conclude that loss of function CBS alleles do not account for a significant proportion of NTDs in Ireland.

Cystathionine beta-Synthase↗
PubMed1997

Promoter-proximal poly(A) sites are processed efficiently, but the RNA products are unstable in the nucleus.

The presence of two polyadenylation signals in the primary transcript of the human immunodeficiency virus type 1 (HIV-1) provirus leads to a requirement for regulation of 3'-end processing. To ensure that viral genome replication and gene expression occur, polyadenylation must occur at the poly(A) site transcribed from the 3' long terminal repeat (LTR) but not the 5' LTR. Models that have been proposed to explain this regulation include (i) inhibition of the 5' site as a result of proximity to the promoter and (ii) enhancement of the 3' site by U3 sequences that are transcribed upstream of only the 3' poly(A) site. In previous studies designed to investigate these models, a reduction in the levels of steady-state RNA was observed when the HIV-1 poly(A) site was placed within 500 nucleotides of the cap site. Although these findings were interpreted to be the result of promoter proximity effects on 3'-end processing, in vitro studies demonstrated that the HIV-1 poly(A) site was equally functional in promoter-proximal and promoter-distal positions. These results led to the hypothesis that, in vivo, the poly(A) site is fully active at this close distance but the short transcripts produced are highly unstable in the nucleus and undergo rapid degradation, precluding their appearance as abundant mRNAs in the steady-state pool. To investigate the biogenesis of these short RNAs in vivo, experiments were performed to examine directly the nuclear processing rates of the HIV-1 poly(A) site in intact cells. By using recombinant adenoviruses as expression vectors, it is now demonstrated conclusively that the HIV-1 poly(A) site is indeed processed at equivalent levels independent of its distance from the promoter. Although transcripts containing the promoter-proximal poly(A) site are processed efficiently, most undergo degradation in the nucleus instead of nucleocytoplasmic transport.

Adenoviridae↗
PubMed1996

Reciprocal effects of splicing and polyadenylation on human immunodeficiency virus type 1 pre-mRNA processing.

Insertion of a functional splicing cassette into a construct containing the HIV-1 poly(A) site followed by the adenovirus L3 poly(A) site results in both specific stimulation of 3' end processing at the HIV-1 site and an increase in the steady-state levels of RNA processed at both sites. To further evaluate this influence of splicing on processing of the HIV-1 poly(A) site, defined mutations which alter or abolish splicing of the intron were made and analyzed for their effects on polyadenylation and steady-state levels of RNA. The data show that a point mutation at the 3' splice site caused activation of a cryptic splice acceptor that is as efficient as the wild-type acceptor. Substitution of this mutant intron for the wild-type intron resulted in stimulation of the HIV-1 poly(A) site to levels equivalent to those caused by the wild-type intron. This mutant did not, however, have as great an effect on steady-state RNA levels as the wild-type intron. A second construct containing a mutated branch point and polypyrimidine tract resulted in abolishment of splicing and a decrease in both poly(A) site use and steady-state levels of RNA. These data demonstrate that the enhanced use of the HIV-1 poly(A) site is a direct result of the splicing reaction, and not merely due to the sequences that were inserted. The effect that poly(A) site strength has on splicing was also addressed. Using activation of the cryptic splice acceptor to indicate changes in splicing efficiency resulting from alterations in poly(A) site strength, it was determined that poly(A) site strength does have an effect on the efficiency of the splicing reaction.

Cell Line, Transformed↗
PubMed1996

Effect of increasing dietary folate on red-cell folate: implications for prevention of neural tube defects.

BACKGROUND: Recommendations by the UK Department of Health suggest that protection from neural tube defects (NTD) can be achieved through intakes of an extra 400 microgram daily of folate/folic acid as natural food, foods fortified with folic acid, or supplements. The assumption is that all three routes of intervention would have equal effects on folate status. METHODS: We assessed the effectiveness of these suggested routes of intervention in optimising folate status. 62 women were recruited from the University staff and students to take part in a 3-month intervention study. Participants were randomly assigned to one of the following five groups: folic acid supplement (400 microgram/day; I); folic-acid-fortified foods (an additional 400 microgram/day; II); dietary folate (an additional 400 microgram/day; III); dietary advice (IV), and control (V). Responses to intervention were assessed as changes in red-cell folate between pre-intervention and post-intervention values. FINDINGS: 41 women completed the intervention study. Red-cell folate concentrations increased significantly over the 3 months in the groups taking folic acid supplements (group I) or food fortified with folic acid (group II) only (p<0.01 for both groups). By contrast, although aggressive intervention with dietary folate (group III) or dietary advice (group IV) significantly increased intake of food folate (p<0.001 and p<0.05, respectively), there was no significant change in folate status. INTERPRETATION: We have shown that compared with supplements and fortified food, consumption of extra folate as natural food folate is relatively ineffective at increasing folate status. We believe that advice to women to consume folate-rich foods as a means to optimise folate status is misleading.

Adolescent↗
PubMed1996

Homocysteine and neural tube defects.

It is now well established that folic acid, when taken periconceptionally, can prevent many neural tube defects. It is also becoming clear that folic acid does not work by correcting a nutritional deficiency in pregnant women. Rather, it appears that a metabolic defect is responsible for these neural tube defects and that this defect or defects can be corrected by a sufficiently large dose of folic acid. Our recent work demonstrates that homocysteine metabolism is likely to be the critical pathway affected by folic acid. We have demonstrated significantly higher homocysteine levels in women carrying affected fetuses than in control women. These findings indicate that one of the enzymes responsible for homocysteine metabolism is likely to be abnormal in affected pregnancies. Animal studies suggest that the conversion of homocysteine to methionine could be the critical step. Rat embryos in culture require methionine for neural tube closure. Methionine synthase, cystathionine synthase, and 5,10 methylene tetrahydrofolate reductase are all important in the metabolism of homocysteine in humans. If methionine synthase is the critical enzyme, it would raise the interesting public health issue that vitamin B-12 might be able to stimulate the abnormal enzyme as folic acid does. Adding vitamin B-12 might make it possible to reduce the dose of folic acid required in fortified food, thus allaying concerns about overexposure to folic acid.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Folate levels and neural tube defects. Implications for prevention.

Using data from a recent case-control study, a woman's risk of having a child with a neural tube defect (NTD) was found to be associated with early pregnancy red cell folate levels in a continuous dose-response relationship. These findings were used to calculate the reduction in NTD cases that would be expected under two different strategies to raise folate levels. Targeting high-risk individuals has a small effect on the population prevalence but can substantially change an individual's risk. Targeting the population produces a small change in individual risk but has a large effect on the population prevalence. Supplementation of high-risk women would be the most efficient method to implement the high-risk strategy, while food fortification would be preferable for the population approach. The current guidelines for the prevention of NTD are for an increased folic acid intake of 0.4 mg per day. This would result in a 48% reduction in NTDs, which may be near optimal. The two intervention strategies should be considered complementary in prevention of NTDs.

Case-Control Studies↗
PubMed1995

Homocysteine metabolism in pregnancies complicated by neural-tube defects.

Folic acid taken around the time of conception can prevent many neural-tube defects. Women with low-normal vitamin B12 values may also be at increased risk. We considered whether homocysteine metabolism via the enzyme methionine synthase, which requires both folate and B12, could be the critical defect in folate-related neural tube defects. Blood was obtained during pregnancies that produced 81 infants with neural-tube defects and 323 normal children. Samples were assayed for homocysteine, methylmalonic acid, plasma folate, red-cell folate, and B12. Mothers of children with neural-tube defects had significantly higher homocysteine values (8.62 [SD 2.8] mumol/L) than did B12-matched controls (7.96 [2.5] mumol/L, p = 0.03). The difference was significant (p = 0.004) in the lower half of the B12 distribution after adjusting for plasma folate. Our study shows that an abnormality in homocysteine metabolism, apparently related to methionine synthase, is present in many women who give birth to children with neural-tube defects. Overcoming this abnormality is likely to be the mechanism by which folic acid prevents neural-tube defects. These findings suggest that the most effective periconceptional prophylaxis to prevent neural-tube defects may require B12 as well as folic acid.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

The biochemical basis of the neuropathy in cobalamin deficiency.

The pathogenesis of the neuropathy associated with vitamin B12 deficiency (subacute combined degeneration (SCD)) is now thought to be related to interference with the methylation reactions in the CNS. The methylation reactions are processed by S-adenosylmethionine (SAM), which is controlled by its product, S-adenosylhomocysteine (SAH). The relationship of these two compounds is termed the methylation ratio. It has been demonstrated that if the ratio falls, the methylation reactions are inhibited leading to a state of CNS hypomethylation. The ratio can fall either due to a rise in SAH or a fall in SAM. It is suggested that for clinical signs to develop in animals who are susceptible to the lesion, both events are usually required. Inhibition of the vitamin B12-dependent enzyme, methionine synthase, leads to a rapid fall in the ratio in the CNS, since unlike other organs such as the liver, it does not have an alternative method of re-methylating homocysteine to maintain the endogenous synthesis of SAM. The supply of methyl groups necessary for the re-methylation reactions is controlled by a series of enzymes, which include methionine synthase. The inborn errors of metabolism that produce deficiency or impairment of these enzymes are described. Neurological syndromes associated with deficiency of these enzymes have close associations with SCD. The other clinical evidence and animal experiments that support this hypothesis are also described.

Animals↗
PubMed1995

An abnormal methylation ratio induces hypomethylation in vitro in the brain of pig and man, but not in rat.

1. The ratio of the methyl donor, S-adenosylmethionine, to the co-product, S-adenosylhomocysteine (the methylation ratio) is known to control the activity of methyltransferases in tissues. Inactivation of the vitamin B12-dependent enzyme, methionine synthase, reduces the methylation ratio in rats and pigs in vivo. 2. We have determined the effect that such alterations have on neural protein 'O' and 'N' methyltransferases using an in vitro assay in rats, pigs and humans in the presence of the normal methylation ratio and the abnormal methylation ratios found experimentally in vivo in rats and pigs. 3. The methylation ratio found in the neural tissues of vitamin B12-inactivated pigs significantly inhibits the protein methyltransferases of pigs and humans. 4. By contrast, the altered methylation ratio found in vitamin B12-inactivated rats only marginally inhibits the equivalent rat methyltransferases. 5. This is consistent with the induction of a myelopathy by such treatment in pigs and humans, but not in the rat. 6. Dietary supplements of methionine given to vitamin B12-inactivated pigs have been shown to prevent the myelopathy in vivo by both elevating the neural S-adenosylmethionine level and resetting the methylation ratio. We find in our in vitro assay that these events reinstate the methyltransferase activity to near normal levels, thus explaining its protective effect in vivo.

Animals↗
PubMed1995

Demonstration of hypomethylation of proteins in the brain of pigs (but not in rats) associated with chronic vitamin B12 inactivation.

1. Pigs treated with nitrous oxide for periods of 1, 2 and 4 months demonstrated markedly reduced levels of methionine synthase and concomitant reduction in the ratio of S-adenosylmethionine to S-adenosylhomocysteine, the methylation ratio, at all time intervals. 2. Both 'O' and 'N' methylations were significantly reduced in pigs after 4 months in nitrous oxide but not after shorter periods. 3. Hypomethylation correlated with the development of clinical ataxia, but was absent when the pigs were clinically normal. It also only occurred when the S-adenosylmethionine level fell. 4. Rats maintained in nitrous oxide for 4 months showed a marked reduction of methionine synthase but no reduction in the methylation ratio or in brain hypomethylation. None of the rats became clinically ataxic. 5. Using an exogenous protein as a methyl group acceptor, it was demonstrated in an in vitro assay that the methyltransferase enzymes responsible for brain 'O' and 'N' methylation were not affected per se by nitrous oxide treatment. 6. It is concluded that reduction of the methylation ratio in the brain of pigs as a consequence of methionine synthase inhibition leads to brain hypomethylation. This hypomethylation could affect critical components of nerve tissue, inducing the vacuolar myelopathic changes seen in the spinal cord of these animals, which mimic those of subacute combined degeneration in man.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Folate catabolism is related to growth rate in weanling rats.

The relationship between growth rate and folate catabolism was determined using a catch-up growth model in weanling rats. A recently developed HPLC method was employed to compare the urinary excretion of the major end product of folate catabolism, acetamidobenzoylglutamate (apABGlu), in normally growing (n = 8) and age-matched growth-restricted (n = 8) weanling animals, during and after food restriction. During the 12-d period of food restriction in which restricted rats were fed (per gram of body weight) at a level of 50% of the required dietary intake for normal growth, apABGlu excretion was significantly higher in normally growing rats compared with growth-restricted rats. In the latter group after restriction, apABGlu concentrations increased markedly, tending to exceed those of normally growing rats of comparable age. Thus growth retardation resulted in a decreased rate of folate catabolism, whereas accelerated growth following dietary restriction was associated with an increase in catabolism. Because early growth in rats is predominantly hyperplastic, these results suggest that the rate of folate catabolism is related to folate utilization in cell division.

4-Aminobenzoic Acid↗
PubMed1995

para-acetamidobenzoylglutamate is a suitable indicator of folate catabolism in rats.

The amount of biologically active folate excreted in the urine corresponds to a small fraction of the recommended dietary allowance, suggesting that a large amount of folate is excreted as its catabolites. A strategy of assessing folate requirement by measuring the daily urinary levels of products of folate catabolism depends on the demonstration of an exclusive mechanism of breakdown as well as a suitable marker of the catabolic process. Rats were given [3H] and [14C]folic acid by gastric intubation daily for 10 d to simulate normal dietary intake of the vitamin. Total urine was collected throughout this period as well as for the following 10 d. Reverse-phase HPLC of the radiolabeled urinary products revealed the presence of a variety of intact folates as well as products of C9-N10 scission of the folate molecule, pteridines, para-aminobenzoylglutamate and para-acetamidobenzoylglutamate. We detected no other N10-containing catabolites, nor did we find the oxidized folate derivative '4 alpha-hydroxy-5-methyltetrahydrofolate'. Of all the urinary folate metabolites, only para-acetamidobenzoylglutmate persisted at high levels up to 10 d after radiolabel treatment was withdrawn. We conclude that folate catabolism occurs exclusively through C9-N10 cleavage and that measurement of urinary para-acetamidobenzoylglutmate provides a suitable indicator of daily folate turnover.

4-Aminobenzoic Acid↗
PubMed1995

Terbutaline vs albuterol for out-of-hospital respiratory distress: randomized, double-blind trial.

OBJECTIVE: To determine the efficacy and safety of single doses of subcutaneous terbutaline (TERB) or nebulized albuterol (ALB) during out-of-hospital treatment for respiratory distress from asthma or chronic obstructive pulmonary disease. METHODS: Patients aged > 18 years who had respiratory distress were enrolled in a double-placebo, double-blind, randomized trial. Paramedics measured respiratory severity using an empiric score [respiratory rate, wheezing, speech, and peak expiratory flow rate (PEFR)], and the patients rated their own respiratory distress using a visual analog scale (VAS). The patients received O2 plus ALB (2.5 mg) and saline injection (n = 40) or TERB (0.25 mg) and saline aerosol (n = 43). RESULTS: The groups were similar with respect to age, gender, initial empiric scores (median score 9 for both groups), PEFRs (89 +/- 84 L/min, mean +/- SD, for ALB vs 97 +/- 84 L/min for TERB), and respiratory distress VAS scores. Both groups showed significant improvement in their respiratory distress VAS scores by the time of ED arrival. The ALB group had a greater improvement in respiratory distress VAS score than did the TERB group (p < 0.05). Empiric scores, PEFR scores, and hospital admission frequencies were not significantly different. No complication was observed. CONCLUSION: The out-of-hospital administration of either aerosolized ALB or subcutaneous TERB reduced respiratory severity. Albuterol provided greater subjective improvement in respiratory distress.

Administration, Inhalation↗
PubMed1995

A genetic defect in 5,10 methylenetetrahydrofolate reductase in neural tube defects.

It is now well-established that folic acid, taken peri-conceptionally, can reduce the risk of neural tube defects (NTDs). Recent work has demonstrated that an abnormality of homocysteine metabolism is a critical factor. The gene for 5,10 methylenetetrahydrofolate reductase, an enzyme important in homocysteine metabolism, was studied in relation to NTDs. To determine the frequency of the allele for the thermolabile form of the reductase, DNA samples were collected from people with NTDs, parents of people with NTDs, and normal controls. Of 82 people with NTDs, 15 (18.3%) were homozygous for the abnormal, thermolabile allele. This was significantly higher (p = 0.01) than the rate of 6.1% in the control population (odds ratio 3.47, 95% CI 1.28-9.41). This is the first specific genetic abnormality to be identified in NTDs. It explains the association between some NTDs and elevated homocysteine, given that the reductase is important in homocysteine metabolism. It also explains how folic acid supplementation prevents some NTDs, by overcoming a partial block in the conversion of 5,10 methylenetetrahydrofolate to 5 methyltetrahydrofolate. Genetic screening could identify women who will require folic acid supplements to reduce their risk of having a child with an NTD.

Case-Control Studies↗
PubMed1995

Cobalt-vitamin B12 deficiency and the activity of methylmalonyl CoA mutase and methionine synthase in cattle.

Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal diet that was very low in Co (12.9-17.6 micrograms Co per kg), or the same diet supplemented with cobalt, for a total of 64 weeks. Vitamin B12 deficiency was induced, as judged by hepatic concentrations of vitamin B12 and plasma concentrations of MMA. However, the activity of holo-methylmalonyl CoA mutase was significantly reduced only in brain. This was reflected in very minor alterations in the tissue concentrations of branched chain- and odd numbered-fatty acids. The activity of holo-methionine synthase was significantly reduced in liver and brain, but there were no consequent alterations in the concentrations of phosphatidyl choline and phosphatidyl ethanolamine. This study confirms that cattle are less susceptible to the effects of cobalt deficiency than sheep, and concludes that prolonged cobalt deficiency had little significant effect on tissue metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗