Biomedical subjects

B Fowler

Publications and source records attributed to B Fowler.

At least 55 records · Page 3Linked to original sources

PubMed1998

Binding of human single chain urokinase to Chinese Hamster Ovary cells and cloning of hamster u-PAR.

The plasminogen activator, urokinase (u-PA), interacts with the u-PA receptor (u-PAR) which results in enhanced plasminogen activation on cell surfaces. The u-PAR is comprised of three homologous domains of approximately 90 amino acids, defined by the pattern of disulfide bonds. Domain 1 (amino acids 1-87) binds the ligand. Within this domain, Y57, and a site between residues 47 and 53, have been suggested as ligand contact points. Intradomain interactions also contribute to the interaction of u-PA and u-PAR. The interaction of u-PA with its receptor exhibits some species specificity. Previous studies have shown that human u-PA does not bind to the murine u-PAR and murine u-PA does not recognize human u-PAR. However, human u-PA does interact with bovine cells with high affinity. To further examine the interaction of the human ligand with the u-PAR of a different species, we characterized the binding of human 125I single chain u-PA (scu-PA) to hamster cells. Chinese Hamster Ovary (CHO) cells bound human scu-PA with high affinity and capacity (Kd = 1.13 +/- 0.8 nM; Bmax = 5.45 +/- 0.98 x 10(4) sites/cell). In ligand blotting with human 125I-scu-PA, major bands migrating with apparent Mr's of 74, 49 and 38 kDa were observed. The cDNA of hamster u-PAR was cloned and a single 1.4 kb mRNA species identified in Northern blots of CHO cell RNA. For comparison, we also cloned u-PAR cDNA from human THP-1 cells. Our human sequence was identical to those published for U937 and endothelial cells. These sequences were aligned with the published sequences for the murine, bovine and rat u-PAR's to obtain a consensus sequence for five species. The cysteine residues could be aligned for all species. Y57, which has been suggested as a ligand contact point was also conserved across species. In addition, 5 of the 7 amino acids between amino acids 47 and 53 were conserved in all species. Gly283, the most likely glycosyl-phosphatidyl inositol attachment site, was also conserved in all species. The conservation of these amino acid residues across all five species, attests to their importance in u-PAR function. In addition, the results of our studies suggest that the hamster may be a useful small animal model for studies of human urokinase function.

Amino Acid Sequence↗

PubMed1998

Evidence that the slowing caused by acute hypoxia is modality dependent.

BACKGROUND: AFM (Additive Factors Method) experiments conducted with visual stimuli suggest that the slowing produced by acute hypoxia is located at the earliest preprocessing stage of information processing and that later stages are unaffected (the bottleneck hypothesis). METHODS: To determine the contribution of degraded visual functioning to slowing, we bypassed this modality and measured reaction time in an AFM paradigm to auditory (Experiment 1) and kinesthetic (Experiment 2) stimuli. In both experiments hypoxia was induced with low oxygen mixtures and arterial blood oxygen saturation (SaO2) was controlled at 65%. Task difficulty was manipulated in Experiment 1 with tones that differed in intensity and in Experiment 2 with lifted cylinders that differed in weight. RESULTS: The results for Experiment 1 showed an interaction between task difficulty and hypoxia, indicating slowing of the preprocessing stage. Slowing was not found in Experiment 2. The absence of slowing in Experiment 2 is surprising and indicates that slowing may be confined to vision and audition and may not involve later, more central, stages. We discuss the need to measure cerebral oxygenation in order to understand the sharp differences between the the bottleneck hypothesis, developed by controlling SaO2, and the more traditional behavioral model which postulates multiple cognitive deficits.

Acute Disease↗

PubMed1997

Biological determinants of P300: the effects of a barbiturate on latency and amplitude.

Polich & Kok (1995) (Biological Psychology, 41, 103-146) have recently argued that P300 is not only sensitive to specific 'cognitive' variables, but also to non-specific biological processes such as arousal. Fluctuations in arousal are said to be indexed by an inverse relationship between latency and amplitude. We tested this hypothesis with a drug that decreases arousal-the barbiturate secobarbital sodium. Twelve subjects performed a visual 80-20% oddball task at two levels of stimulus quality and after ingesting the drug (2.9 mg/kg body weight) or a placebo. Reaction time (RT) and P300 were collected simultaneously and the latter was analyzed on both a single trial and average basis. The RT results confirmed that secobarbital interacts with stimulus quality. Secobarbital slowed single trial P300 by about half as much as RT, and this slowing was additive with stimulus quality. Thus the two measures dissociated. Secobarbital did not influence P300 amplitude. Average P300 revealed the same pattern of results, although the size of the latency effects was somewhat attenuated. RT and P300 latency were more strongly correlated than P300 latency and amplitude. We propose that P300 latency reflected the slowing of stimulus evaluation produced by the depressant properties of the drug, and that fluctuations in arousal are not necessarily associated with a simple inverse relationship between P300 latency and amplitude.

Adult↗

PubMed1997

The semiconductor elements arsenic and indium induce apoptosis in rat thymocytes.

Indium arsenide and gallium arsenide are important new materials in the semiconductor industry due to their superior electronic properties in comparison with the older silicon-based materials. Animal experiments have shown that exposure to these compounds induces marked alterations in gene expression and immune response. Toxicity to the immune system has frequently been related to T and B cell apoptosis. In the present study we show that the semiconductor elements indium (In) and arsenic (As) are able to induce apoptosis in rat thymocytes in vitro. The results show that exposure to InCl3 (1, 10, or 100 microM) or Na AsO2 (0.01, 0.1, or 1 microM) induced DNA laddering after 6 h of incubation without compromising cell viability. These results were corroborated by flow cytometry analysis of propidium iodide-loaded cells, showing a typical high hypodiploid DNA peak in apoptotic thymocytes. Higher doses of In (1 mM) or As (10-100 microM) induced cell death by necrosis. These data indicate that In and As can induce apoptosis and necrosis in T lymphocytes in a dose-dependent manner, which may be of relevance for their immunotoxicity.

Animals↗

PubMed1997

Methionine synthase deficiency without megaloblastic anaemia.

UNLABELLED: We report findings on a child presenting with neonatal homocystinuria, hypomethioninaemia and severe neurological symptoms, including developmental delay and seizures. Methylmalonic aciduria was not present. The activity of methionine synthase in fibroblasts was severely deficient and formation of methylcobalamin from 57Co labelled cyanocobalamin was very low. The patients cells complemented with those of a cblE patient but not with those of two cblG patients. No biochemical or clinical response to injections of hydroxycobalamin was found. Both off treatment and on betaine and methionine supplementation the patient, at age 8 years, has not developed megaloblastic anaemia. In addition, the patient is homozygous for the C677T polymorphism in the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene and the concomitant existence of this mutation with the methionine synthase defect may prevent folate < > and thus anaemia. CONCLUSION: We report the lack of megaloblastic anaemia in a patient with severe methionine synthase deficiency who is also homozygous for C677T in MTHFR, hypothesize that the MTHFR polymorphism protects the patient against anaemia and speculate that homozygosity for MTHFR C677T could cause the dissociation between haematological and neurological disease seen in some patients with vitamin B12 deficiency.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Use of nitrous oxide to dissociate the non-specific and specific components of the human auditory N1.

The components of the N1 are thought to be related to sensory functioning (Components 1 and 2) and arousal (Component 3). To provide direct evidence for the involvement of Component 3 in arousal, we hypothesized that it should be more sensitive to the anesthetic gas nitrous oxide (N2O) than Component 1. Using the technique of selective adaptation, 30 blocks of 5 tones were presented at 1 min intervals to 9 subjects who breathed air, 25% and 35% N2O. As hypothesized, the amplitude of Component 3 was significantly reduced in a dose-dependent manner by N2O, but the amplitude of Component 1 was not, although the latter showed some evidence of a decrease at 25% N2O.

Adult↗

PubMed1997

Disorders of homocysteine metabolism.

This review of recent advances covers (1) the metabolism of methionine and its regulation, emphasizing interactions with the three important vitamins folate, cobalamin and pyridoxine; (2) present knowledge of enzymological and moleculargenetic aspects of homozygous deficiencies of the three enzymes which cause elevated homocyst(e)ine; (3) recent clinical findings, post-methionine loading results related to enzyme and mutation studies in obligate heterozygotes for cystathionine beta-synthase deficiency; (4) important new evidence for disturbed homocysteine metabolism in neural tube defects, particularly based on studies of the thermolabile methylene-tetrahydrofolate reductase mutation which is also of importance in vascular disease; (5) the suitability and limitations of animal models that have so far been described.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

PubMed1997

Folate-responsive homocystinuria and megaloblastic anaemia in a female patient with functional methionine synthase deficiency (cblE disease).

This first detailed report of a female patient with functional methionine synthase deficiency due to the cblE defect describes treatment with several vitamins and cofactors and clinical progress for 17 years. Before treatment, major findings were microcephaly, psychomotor retardation, episodic reduced consciousness, megaloblastic anaemia, increased plasma free homocystine (> 20 mumol/L), low plasma methionine (< 10 mumol/L) and increased excretion of formiminoglutamate. On high-dose folic acid, biochemical abnormalities such as formiminoglutamate excretion and homocystinuria nearly normalized, but clinical and haematological abnormalities remained. On replacement of folate with methylcobalamin, alertness, motor function, speech and the electroencephalogram improved, biochemical features were similar, but the mean corpuscular volume increased. The best control was observed on a combination of folate and methylcobalamin. At 17 years of age she remains severely mentally retarded. In cultured fibroblasts methionine synthesis was reduced (0.03 nmol/mg/per 16 h, controls 2.4-6.9); methionine synthase activity was normal under high reducing conditions but decreased on limiting the reducing agent, dithiothreitol, to 5 mmol/L (18% of total, controls 51-81%); formation of methylcobalamin was low (4.5% of total cobalamins, control 57.5%) and complementation studies indicated the cblE defect. Methionine formation showed only minor increases in cells grown in folate- or cobalamin-supplemented medium. Serine synthesis, which was low in normal medium, increased with cobalamin supplementation. These studies suggest further heterogeneity within cblE mutants, show the difficulty of establishing the enzyme defect in vitro, and indicate a role for folate in addition to cobalamin in treatment.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Methionine and serine formation in control and mutant human cultured fibroblasts: evidence for methyl trapping and characterization of remethylation defects.

The conversion of labeled formate to methionine and serine, as a measure of remethylation of homocysteine to methionine and folate coenzyme cycling, has been studied in control and mutant human fibroblasts. Fibroblasts in monolayer culture were incubated with [14C]formate, and labeled methionine sulfone and serine were determined in hydrolysates of oxidized cell proteins. In control cells, methionine and serine were clearly measurable (n = 21, 1.7-5.5 and 2.4-9.7 nmol/mg protein/16 h, respectively). In contrast, methionine formation was reduced in cells from patients with methylenetetrahydrofolate reductase (MR) deficiency (MR mutant, n = 11, 0.05-0.44), combined methylmalonic aciduria/homocystinuria [cobalamin(cbl)C/D mutant, n = 12, 0.014-0.13), and methionine synthase deficiency (MS mutant, n = 3, 0.04-0.23). Furthermore, serine formation was low in cblC/D mutant (0.08-0.98) and MS mutant (0.17-0.94) cells, but normal or high in MR mutant cells (5.2-11.4). Growth of cblC/D mutant cells in medium supplemented with high concentrations of hydroxo-cbl resulted in significant increases of both methionine and serine formation. Taken together these findings provide clear evidence for the existence of the formate to serine pathway described by W. B. Strong and V. Schirch in cultured fibroblasts and indicate that disturbed MS function due to a specific genetic disorder is associated with reduced serine formation in vitro, which reflects availability of reduced folate coenzymes. The correction of this defect by vitamin B12 alone, in cblC/D mutant cell lines, correlates well with the clinical response in the patients and fits in well with the idea that reduced availability of folate coenzymes occurs in functional MS deficiency, in agreement with the methyl trap hypothesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Five patients with a biotin-responsive defect in holocarboxylase formation: evaluation of responsiveness to biotin therapy in vivo and comparative biochemical studies in vitro.

Biochemical studies in five patients with a defect in biotin-responsive holocarboxylase synthesis are reported. The age of onset (2 d to 6 y) as well as the severity of illness varied considerably. In all patients diagnosis was established by the finding of organic aciduria typical for multiple carboxylase deficiency in a catabolic state. In four patients the response to biotin therapy was evaluated by measurement of mitochondrial carboxylase activities in lymphocytes and by monitoring urinary organic acid excretion. In three patients clinical symptoms disappeared with 10-20 mg biotin/d, whereas normalization of the biochemical parameters required higher doses (20-40 mg/d). The fourth patient required a dose of 100 mg biotin/d before her skin rash disappeared. She remains mentally retarded and shows slightly elevated urinary organic acid excretion. Carboxylase activities were clearly deficient in fibroblasts grown in the commonly used medium which contains 10 nmol/L biotin (contributed by FCS in medium) in two patients. Fibroblasts of the other three patients became deficient only in a low biotin medium (0.1 nmol/L). Reactivation of deficient carboxylase activities in relation to time and biotin concentration correlated well with the severity and age of onset of illness in four patients. In one patient, however, carboxylase reactivation followed a more complex pattern requiring the longest incubation time but only a moderately increased biotin concentration of 19 nmol/L compared with 3-5 nmol/L in normal cells and 34-4000 nmol/L in the other four patients. The results in the five patients are in accordance with a primary defect of holocarboxylase synthetase due to a decreased affinity for biotin, in one patient combined with a decreased Vmax.

Acetyl-CoA Carboxylase↗

PubMed1997

Performance during positive pressure breathing after rapid decompression up to 72,000 feet.

Positive pressure breathing (PPB) is a survival system that delivers high-pressure oxygen and body counterpressure in the event of cockpit depressurization at high altitude, but the ability of aircrew to perform an emergency "get-me-down" maneuver with this system is unknown. To address this question, a serial choice reaction time (RT) task was administered to six participants, who were sustained with PPB after rapid decompression to 60,000 feet (18,288 m) or 72,000 feet (21,946 m) in a hypobaric chamber (Experiment 1). Hypoxia was monitored by blood oxygen saturation (SaO2). Experiment 2 assessed the effects of PPB alone on performance. In Experiment 3, performance was measured while the rate of decrease of SaO2 observed in Experiment 1 was replicated with low oxygen mixtures at ground level. During the first 60 s at 72,000 feet, RT increased by 79 ms because of a shift of the whole distribution to the right. PPB at ground level did not increase RT. Decreasing SaO2 at ground level increased RT, but only after a delay. We conclude that inattention to the task, PPB alone, or, surprisingly, hypoxia cannot explain the majority of the impairment observed in Experiment 1. Rather, PPB and low pressure at high altitude interacted to exacerbate lacrimation, thereby degrading vision. Implications for the design of get-me-down maneuvers are discussed.

Adult↗

PubMed1997

The clinical phenotype of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric aciduria): case reports of 23 new patients.

OBJECTIVES: To further define the clinical spectrum of the disease for pediatric and metabolic specialists, and to suggest that the general pediatrician and pediatric neurologist consider succinic semialdehyde dehydrogenase (SSADH) deficiency in the differential diagnosis of patients with (idiopathic) mental retardation and emphasize the need for accurate, quantitative organic acid analysis in such patients. PATIENTS: The clinical features of 23 patients (20 families) with SSADH deficiency (4-hydroxybutyric acid-uria) are presented. The age at diagnosis ranged from 3 months to 25 years in the 11 male and 12 female patients; consanguinity was noted in 39% of families. OUTCOME MEASUREMENTS: The following abnormalities were observed (frequency in 23 patients): motor delay, including fine-motor skills, 78%; language delay, 78%; hypotonia, 74%; mental delay, 74%; seizures, 48%; decreased or absent reflexes, 39%; ataxia, 30%; behavioral problems, 30%; hyperkinesis, 30%; neonatal problems, 26%; and electroencephalographic abnormalities, 26%. Associated findings included psychoses, cranial magnetic resonance or computed tomographic abnormalities, and ocular problems in 22% or less of patients. Therapy with vigabatrin proved beneficial to varying degrees in 35% of the patients. Normal early development was noted in 30% of patients. CONCLUSIONS: Our data imply that two groups of patients with SSADH deficiency exist, differentiated by the course of early development. Our recommendation would be that accurate, quantitative organic acid analysis in an appropriate specialist laboratory be requested for any patients presenting with two or more features of mental, motor, or language delay and hypotonia of unknown cause. Such analyses are the only definitive way to diagnose SSADH deficiency; the diagnosis can be confirmed by determination of enzyme activity in white cells from whole blood. We think that increased use of organic acid determination will lead to increased diagnosis of SSADH deficiency and a more accurate representation of disease frequency. As additional patients are identified, we should have a better understanding of both the metabolic and clinical profiles of SSADH deficiency.

Adolescent↗

PubMed1997

Influence of oral S-adenosylmethionine on plasma 5-methyltetrahydrofolate, S-adenosylhomocysteine, homocysteine and methionine in healthy humans.

Elevated plasma homocysteine concentration is an independent risk factor for vascular disease in humans. In addition to nutritional and genetic factors, an interruption of the coordinate regulatory function of S-adenosylmethionine has been proposed to be involved in the occurrence of hyperhomocysteinemia. The effect of oral S-adenosylmethionine on homocysteine metabolism in humans is unknown. We investigated the effect of oral S-adenosylmethionine (400 mg) on plasma levels of 5-methyltetrahydrofolate, which is the active form of folate in the remethylation of homocysteine to methionine, S-adenosylhomocysteine, the demethylated product of S-adenosylmethionine, homocysteine and methionine over 24 hr in 14 healthy subjects. After oral administration, S-adenosylmethionine increased from 38.0 +/- 13.4 to 361.8 +/- 66.4 nmol/liter (mean +/- S.E., P < .001) and returned to base-line values with a half-life of 1.7 +/- 0.3 hr. Both S-adenosylhomocysteine and 5-methyltetrahydrofolate showed a significant transient increase (from 29.9 +/- 3.7 to 51.7 +/- 7.1 nmol/liter, and from 25.1 +/- 2.5 to 36.2 +/- 3.5 nmol/liter, respectively, P < .001), although homocysteine and methionine did not change over the time of measurement. These changes were not found in subjects without previous S-adenosylmethionine administration. The observed metabolic changes suggest that S-adenosylmethionine, at least in concentrations obtained in this study, does not inhibit 5,10-methylenetetrahydrofolate reductase, the 5-methyltetrahydrofolate forming enzyme. Rather they indicate a positive effect on 5-methyltetrahydrofolate, a key cofactor in homocysteine metabolism, which should be considered in homocysteine lowering strategies for the prevention of vascular disease.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

PubMed1997

Slowing due to acute hypoxia originates early in the visual system.

BACKGROUND: Experiments in the visual modality show that acute hypoxia slows the earliest stage of information processing-preprocessing. It is unknown, however, whether a later stage, feature extraction, is also slowed. METHODS: To answer this question, an additive factors method (AFM) experiment was conducted which employed seven well trained subjects whose arterial oxyhaemoglobin saturation was controlled at 63% with low oxygen mixtures. The subjects responded to oddball names presented on a computer screen and both reaction time (RT) and the event-related brain potential P300 were measured. The luminance and quality of the names was varied factorially to influence the preprocessing and feature extraction stages, respectively. RESULTS: RT and P300 latency showed the same pattern of results: stimulus luminance and signal quality were additive, indicating that AFM assumptions were met; hypoxia and stimulus luminance were interactive but hypoxia and signal quality were additive. CONCLUSION: In conjunction with other evidence, we interpret these results to indicate that the locus of slowing produced by hypoxia is largely at the preprocessing stage, at least in the visual modality. Slowing at the preprocessing stage can be explained by hypoxia shifting the function relating RT and stimulus luminance to the right by a constant amount.

Acute Disease↗

PubMed1996

Coexistence of hereditary homocystinuria and factor V Leiden--effect on thrombosis.

BACKGROUND: Venous and arterial thromboembolism occurs in only about one third of patients homozygous for homocystinuria, which suggests that other, contributory factors are necessary for the development of thrombosis in these patients. Factor V Leiden, an R506Q mutation in the gene coding for factor V, is the most common cause of familial thrombosis and could be a potentiating factor. METHODS: We determined activated partial-thromboplastin times in the presence and absence of activated protein C and tested for the factor V Leiden mutation in 45 members of seven unrelated consanguineous kindreds in which at least 1 member was homozygous for homocystinuria. RESULT: Thrombosis (venous, arterial, or both) occurred in 6 of 11 patients with homocystinuria (age, 0.2 to 8 years). All six also had the factor V Leiden mutation. One patient with prenatally diagnosed homocystinuria who was also heterozygous for factor V Leiden has received warfarin therapy since birth and has not had thrombosis (age, 18 months). Of four patients with homocystinuria who did not have factor V Leiden, none had thrombosis (ages at this writing, 1 to 17 years). Three women who were heterozygous for both homocystinuria and factor V Leiden had recurrent fetal loss and placental infarctions. CONCLUSIONS: Patients with concurrent homocystinuria and factor V Leiden can have an increased risk of thrombosis. Screening for factor V Leiden may be indicated in patient with homocystinuria and their family members.

Adolescent↗

PubMed1996

Effect of methionine loading on 5-methyltetrahydrofolate, S-adenosylmethionine and S-adenosylhomocysteine in plasma of healthy humans.

1. Elevated plasma homocysteine concentration, either in the fasting state or after methionine loading, is an independent risk factor for vascular disease in man. Methionine loading has been used to investigate impaired methionine metabolism, especially of the trans-sulphuration pathway, but most studies have focused on changes in homocysteine. 2. We investigated the effect of methionine excess on total plasma homocysteine, 5-methyltetrahydrofolate (which is the active form of folate in the remethylation of homocysteine to methionine), S-adenosyl-methionine (the first metabolite of methionine) and S-adenosylmethionine) (the demethylated product of S-adenosylmethionine) over 24h in 12 healthy subjects. 3. As well as the expected increase in homocysteine (from 8.0 +/- 1.3 to 32.6 +/- 10.3 mumol/l, mean +/- SD, P < 0.001), S-adenosylmethionine showed a significant transient increase (from 37.9 +/- 25.0 to 240.3 +/- 109.2 nmol/l, P < 0.001), which correlated well with homocysteine (r2 = 0.92, P < 0.001). 5-Methyltetrahydrofolate values decreased significantly (from 23.2 +/- 7.2 to 13.1 +/- 2.9 nmol/l, P < 0.01), and gradually returned to baseline levels after 24h. No significant change over the time of measurement was found for S-adenosylhomocysteine. 4. The sequence of metabolic changes observed in this study strongly suggests that a change in either homocysteine or S-adenosylmethionine may cause a reduction in 5-methyltetrahydrofolate. This must be considered in evaluating the relationship between folate and homocysteine in vascular disease. The metabolic relationships illustrated in this study should be evaluated in the search for pathogenetic mechanisms of mild hyperhomocysteinaemia and vascular disease.

Adult↗

PubMed1996

An AFM investigation of the effects of acute hypoxia on mental rotation.

To determine if the stimulus identification stage of information processing is slowed by hypoxia, eight highly trained subjects performed a mental rotation task involving same or different responses to pairs of two-dimensional figures rotated in the picture plane. They breathed low oxygen mixtures adjusted to maintain arterial blood oxygen saturation at 64%. The results showed that hypoxia increased the intercept rather than the slope of the linear function relating reaction time to angle of rotation. According to Additive Factors Method logic, this result indicates that hypoxia does not slow stimulus identification. This is further evidence against the long-held view that hypoxia impairs information processing mechanisms throughout the system--the multiple loci hypothesis. The authors propose instead the early slowing hypothesis. According to this view, the slowing produced by hypoxia is highly specific to one or two early stages of visual processing, and this early slowing acts as a bottleneck to later processing.

Adult↗

PubMed1996

Defects in human methionine synthase in cblG patients.

Inborn errors resulting in isolated functional methionine synthase deficiency fall into two complementation groups, cblG and cblE. Using biochemical approaches we demonstrate that one cblG patient has greatly reduced levels of methionine synthase while in another, the enzyme is specifically impaired in the reductive activation cycle. The biochemical data suggested that low levels of methionine synthase activity in the first patient may result from mutations in the catalytic domains of the enzyme, reduced transcription, or generation of unstable message or protein. Using Northern analysis, we demonstrate that the molecular basis for the biochemical phenotype in this patient is associated with greatly diminished steady-state levels of methionine synthase mRNA. The biochemical data on the second patient cell line implicated mutations specific to reductive activation, a function that is housed in the C-terminal AdoMet-binding domain and the intermediate B12-binding domain, in the highly homologous bacterial enzyme. We have detected two mutations in a compound heterozygous state, one that results in conversion of a conserved proline (1173) to a leucine residue and the other a deletion of an isoleucine residue (881). The crystal structure of the C-terminal domain of the Escherichia coli MS predicts that the Pro to Leu mutation could disrupt activation since it is embedded in a sequence that makes direct contacts with the bound AdoMet. Deletion of isoleucine in the B12-binding domain would result in shortening of a beta-sheet. Our data provide the first evidence for mutations in the methionine synthase gene being culpable for the cblG phenotype. In addition, they suggest directly that mutations in methionine synthase can lead to elevated homocysteine, implicated both in neural tube defects and in cardiovascular diseases.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗