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Biomedical subjects

R A Gravel

Publications and source records attributed to R A Gravel.

At least 19 recordsLinked to original sources
PubMed2006

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway.

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

Animals↗
PubMed2003

Widespread distribution of beta-hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol.

Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the beta-subunit of hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G(M2) ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the beta-subunit of beta-hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.

Adenoviridae↗
PubMed2000

Expression in Escherichia coli of N- and C-terminally deleted human holocarboxylase synthetase. Influence of the N-terminus on biotinylation and identification of a minimum functional protein.

Biotin functions as a covalently bound cofactor of biotindependent carboxylases. Biotin attachment is catalyzed by biotin protein ligases, called holocarboxylase synthetase in mammals and BirA in prokaryotes. These enzymes show a high degree of sequence similarity in their biotinylation domains but differ markedly in the length and sequence of their N terminus. BirA is also the repressor of the biotin operon, and its DNA attachment site is located in its N terminus. The function of the eukaryotic N terminus is unknown. Holocarboxylase synthetase with N- and C-terminal deletions were evaluated for the ability to catalyze biotinylation after expression in Escherichia coli using bacterial and human acceptor substrates. We showed that the minimum functional protein is comprised of the last 349 of the 726-residue protein, which includes the biotinylation domain. Significantly, enzyme containing intermediate length, N-terminal deletions interfered with biotin transfer and interaction with different peptide acceptor substrates. We propose that the N terminus of holocarboxylase synthetase contributes to biotinylation through N- and C-terminal interactions and may affect acceptor substrate recognition. Our findings provide a rationale for the biotin responsiveness of patients with point mutations in the N-terminal sequence of holocarboxylase synthetase. Such mutant enzyme may respond to biotin-mediated stabilization of the substrate-bound complex.

Base Sequence↗
PubMed2000

A common polymorphism in methionine synthase reductase increases risk of premature coronary artery disease.

BACKGROUND: Methionine synthase reductase (MTRR) catalyzes the regeneration of methylcobalamin, a cofactor of methionine synthase, an enzyme essential for maintaining adequate intracellular pools of methionine and tetrahydrofolate, as well as for maintaining homocysteine concentrations at nontoxic levels. We recently identified a common A-->G polymorphism at position 66 of the cDNA sequence of MTRR; this variant was associated with a greater than normal risk for spina bifida in the presence of low levels of cobalamin. OBJECTIVE: To investigate whether the polymorphism was associated with alterations in levels of homocysteine, folate, and vitamin B12, and with risk of developing premature coronary artery disease (CAD), in a population of individuals presenting for cardiac catheterization procedures. METHODS: We screened 180 individuals aged < 58 years with angiographically documented coronary-artery occlusions or occlusion-free major arteries for the presence of the 66A-->G MTRR polymorphism using a polymerase-chain-reaction-based assay. RESULTS: We identified a trend in risk of premature CAD across the genotype groups (P = 0.03) with a sex-adjusted relative risk of premature CAD equal to 1.49 (95% confidence interval 1.10-2.03) for the GG versus AA genotype groups. There was no difference in fasting levels of plasma total homocysteine, serum folate, and vitamin B12 among the three MTRR genotypes. CONCLUSIONS: Our findings suggest that the GG genotype of MTRR is a significant risk factor for the development of premature CAD, by a mechanism independent of the detrimental vascular effects of hyperhomocysteinemia. This association needs to be confirmed in other studies.

Age Factors↗
PubMed1999

Molecular cloning, expression and physical mapping of the human methionine synthase reductase gene.

Methionine synthase reductase (EC 2.1.1.135) is a flavoprotein essential for maintenance of methionine synthase in an active state. We characterized the human gene for methionine synthase reductase (MTRR). The gene is approximately 34kb and comprises 15 exons, varying in size from 43 to 1213bp, and 14 introns whose sizes vary from 108bp to 5kb. The positions of several junctions are conserved between the MTRR gene and the C. elegans ortholog, as well as with the rat cytochrome P450 reductase gene. A 1.3kb CpG island encompasses the 5'-flanking region and exon 1 and extends into intron 1. A short region including the transcription start site is sufficient to confer promoter activity, with a better outcome when accompanied by intron 1. The promoter region contains putative binding sites for Sp1, AP-1, AP-2 as well as CAAT motifs, but no consensus TATA box. Primer extension analysis revealed a single major transcription start site, located 137bp upstream of the previously reported initiator ATG. An alternative splicing event involving a portion of exon 1 predicts that translation can potentially be initiated at two different ATG codons. The gene was physically assigned to a narrow area between markers WI1755 and D5S1957.

Animals↗
PubMed1999

Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects.

Folic acid administration to women in the periconceptional period reduces the occurrence of neural tube defects (NTDs) in their offspring. A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), 677C-->T, is the first genetic risk factor for NTDs in man identified at the molecular level. The gene encoding another folate-dependent enzyme, methionine synthase (MTR), has recently been cloned and a common variant, 2756A-->G, has been identified. We assessed genotypes and folate status in 56 patients with spina bifida, 62 mothers of patients, 97 children without NTDs (controls), and 90 mothers of controls, to determine the impact of these factors on NTD risk. Twenty percent of cases and 18% of case mothers were homozygous for the MTHFR polymorphism, compared to 11% of controls and 11% of control mothers, indicating that the mutant genotype conferred an increased risk for NTDs. The risk was further increased if both mother and child had this genotype. The MTR polymorphism was associated with a decreased O.R. (O.R.); none of the cases and only 10% of controls were homozygous for this variant. Red blood cell (RBC) folate was lower in cases and in case mothers, compared to their respective controls. Having a RBC folate in the lowest quartile of the control distribution was associated with an O.R. of 2.56 (95% CI 1.28-5.13) for being a case and of 3.05 (95% CI 1.54-6.03) for being a case mother. The combination of homozygous mutant MTHFR genotype and RBC folate in the lowest quartile conferred an O.R. for being a NTD case of 13.43 (CI 2.49-72.33) and an O.R. for having a child with NTD of 3.28 (CI 0.84-12.85). We propose that the genetic-nutrient interaction--MTHFR polymorphism and low folate status--is associated with a greater risk for NTDs than either variable alone.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Overview of mutations in the PCCA and PCCB genes causing propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl-CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd-numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of alpha and beta subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of alpha and beta subunits by Western blot of many PCCA patients, as well as conventional mRNA-minus result of Northern blots for either gene or beta subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT-PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene-no mutation is predominant in the populations studied-while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Alternative Splicing↗
PubMed1999

Mechanism of biotin responsiveness in biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans. A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency. We have evaluated the biotin responsiveness associated with six missense mutations previously identified in affected patients by expression of plasmids containing the mutated HCS in an Escherichia coli strain mutated in the corresponding BirA gene. We demonstrate that the mutations identified in the MCD patients are indeed responsible for their reduced HCS activity. Four of the mutations, clustering in the putative biotin binding domain as deduced from the structure of the E. coli enzyme, are consistent with an explanation for biotin responsiveness based on altered affinity for biotin. The remaining mutations, located outside the biotin binding region, were associated with a more limited biotin responsiveness that may be explained by the degree of residual enzyme activity present. The data suggest that the concentration of circulating biotin is as low as 100 times below the Km of the enzyme, so that any increase in biotin concentration through dietary supplementation would result in saturation of the available mutant enzyme. We suggest that these alternative explanations are sufficient to account for the apparent universality of biotin responsiveness in biotin responsive multiple carboxylase deficiency.

Bacterial Proteins↗
PubMed1999

A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida.

Impairment of folate and cobalamin (vitamin B(12)) metabolism has been observed in families with neural tube defects (NTDs). Genetic variants of enzymes in the homocysteine remethylation pathway might act as predisposing factors contributing to NTD risk. The first polymorphism linked to increased NTD risk was the 677C-->T mutation in methylenetetrahydrofolate reductase (MTHFR). We now report a polymorphism in methionine synthase reductase (MTRR), the enzyme that activates cobalamin-dependent methionine synthase. This polymorphorism, 66A-->G (I22M), has an allele frequency of 0.51 and increases NTD risk when cobalamin status is low or when the MTHFR mutant genotype is present. Genotypes and cobalamin status were assessed in 56 patients with spina bifida, 58 mothers of patients, 97 control children, and 89 mothers of controls. Cases and case mothers were almost twice as likely to possess the homozygous mutant genotype when compared to controls, but this difference was not statistically significant. However, when combined with low levels of cobalamin, the risk for mothers increased nearly five times (odds ratio (OR) = 4.8, 95% CI 1.5-15.8); the OR for children with this combination was 2.5 (95% CI 0.63-9.7). In the presence of combined MTHFR and MTRR homozygous mutant genotypes, children and mothers had a fourfold and threefold increase in risk, respectively (OR = 4.1, 95% CI 1.0-16.4; and OR = 2.9, 95% CI 0.58-14.8). This study provides the first genetic link between vitamin B(12) deficiency and NTDs and supports the multifactorial origins of these common birth defects. Investigation of this polymorphism in other disorders associated with altered homocysteine metabolism, such as vascular disease, is clearly warranted.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Detection of a normally rare transcript in propionic acidemia patients with mRNA destabilizing mutations in the PCCA gene.

Propionic acidemia is an autosomal recessive disorder caused by a deficiency in the mitochondrial enzyme propionyl-CoA carboxylase (PCC). PCC is composed of two subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. We analyzed mutations of the PCCA gene using patients' fibroblasts diagnosed with alpha subunit deficiency. By RT-PCR, four of 12 cell lines examined appeared to have a larger transcript present at a level comparable with that of the expected normal species. Sequencing of the larger transcriptrevealed an 84 bp insertion at nt 1209 of the codingsequence. Its incorporation in the transcript results in translation termination due to the presence of two in-frame stop codons. The 84 bp insertion was found to originate from the intron between nt 1209 and 1210. Consensus splice donor and acceptor sites were found at the 3'- and 5'-ends of the insertion, respectively. The insertion was also found in the remaining eight cell lines as well as in normal cells, but at a muchreduced level compared with the normal lengthsequence. Mutation analysis of the four cell lines showing seemingly elevated levels of the insertion sequence revealed one nonsense mutation (R288X), two frameshift deletions (700del5 and 1115del4) and one splice mutation (1671IVS+5G-->C) as expressed alleles. We conclude that the common characteristic of the four cell lines is that they contain mRNA destabilizing mutations that reduce the mRNA level of the normal length sequence. Consequently, the low levels of cryptic mRNAs become detectable at a level similar to that of the residual level of the normal length mRNA. We suggest that screening for an increased proportion of the 84 bp insertion by RT-PCR can be used as a rapid assay for RNA destabilizing mutations. Our results suggest caution in associating such mutations with aberrant mRNA species, such as cryptic splice products, which may instead be part of the 'background noise' of the splicing machinery.

Base Sequence↗
PubMed1999

Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism.

Methionine synthase reductase (MSR) deficiency is an autosomal recessive disorder of folate/cobalamin metabolism leading to hyperhomocysteinemia, hypo- methioninemia and megaloblastic anemia. Deficiency in MSR activity occurs as the result of a defect in the MSR enzyme, which is required for the reductive activation of methionine synthase (MS). MS itself is responsible for the folate/cobalamin-dependent conversion of homo- cysteine to methionine. We have recently cloned the cDNA corresponding to the MSR protein, a novel member of the ferredoxin-NADP(+)reductase (FNR) family of electron transferases. We have used RT-PCR, heteroduplex, single-strand conformation poly- morphism (SSCP) and DNA sequence analyses to reveal 11 mutations in eight patients from seven families belonging to the cblE complementation group of patients of cobalamin metabolism that is defective in the MSR protein. The mutations include splicing defects leading to large insertions or deletions, as well as a number of smaller deletions and point mutations. Apart from an intronic substitution found in two unrelated patients, the mutations appear singular among individuals. Of the eleven, three are nonsense mutations, allowing for the identification of two patients for whom little if any MSR protein should be produced. The remaining eight involve point mutations or in-frame disruptions of the coding sequence and are distributed throughout the coding region, including proposed FMN, FAD and NADPH binding sites. These data demonstrate a unique requirement for MSR in the reductive activation of MS.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells.

Tay-Sachs disease is a severe, inherited disease of the nervous system caused by accumulation of the brain lipid GM2 ganglioside. Mouse models of Tay-Sachs disease have revealed a metabolic bypass of the genetic defect based on the more potent activity of the enzyme sialidase towards GM2. To determine whether increasing the level of sialidase would produce a similar effect in human Tay-Sachs cells, we introduced a human sialidase cDNA into neuroglia cells derived from a Tay-Sachs fetus and demonstrated a dramatic reduction in the accumulated GM2. This outcome confirmed the reversibility of GM2 accumulation and opens the way to pharmacological induction or activation of sialidase for the treatment of human Tay-Sachs disease.

Base Sequence↗
PubMed1999

Neuronal apoptosis in sudden infant death syndrome.

Although evidence shows that victims of sudden infant death syndrome (SIDS) suffer repetitive episodes of hypoxemia, only subtle abnormalities have been found in their brains by light microscopy. The aim of the present study was to determine whether apoptosis, a form of cell death that can be triggered by hypoxemia and that leaves no scarring detectable by light microscopy, would be present in hypoxia-sensitive brain regions of SIDS victims. We looked for the presence of apoptosis with an in situ end-labeling method that detects DNA fragmentation. We studied 29 SIDS victims who were age-matched to nine control cases. We found significant neuronal apoptosis in 79% of the SIDS cases: 55% of the cases positive in the hippocampus and 96% positive in the brainstem. Whereas the distribution of apoptosis in the hippocampus was in hypoxia-sensitive subregions, the distribution in the brainstem was mostly in dorsal nuclei, including those involved with sensation in the face and position of the head (nucleus of the spinal trigeminal tract and vestibular nuclei). The control cases showed no significant apoptosis in the hippocampus and a mild degree in the brainstem in three cases. Our results indicate the occurrence of an acute insult at least several hours before death, an insult from which the infants had apparently recuperated. This suggests that SIDS victims suffered repeated apoptosis resulting in significant neuronal damage and, thus, functional loss in key brain regions. The involvement of specific nuclei in the brainstem may be linked to the fact that prone sleeping is a significant risk factor for SIDS. Enhanced neuronal death by apoptosis may thus have major implications for understanding the sequence of events leading to SIDS.

Apoptosis↗
PubMed1998

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria.

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1998

A frequent TG deletion near the polyadenylation signal of the human HEXB gene: occurrence of an irregular DNA structure and conserved nucleotide sequence motif in the 3' untranslated region.

While screening for new mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase, a TG deletion (deltaTG) was found in the 3' untranslated region (3'UTR) of the gene, 7 bp upstream from the polyadenylation signal. Examination of DNA samples of 145 unrelated Argentinean individuals from different racial backgrounds showed that the deltaTG allele was present with a frequency of approximately 0.1, compared with the wild-type (WT) allele. The deletion was not associated with infantile or variant forms of Sandhoff disease when present in combination with a deleterious allele. Total Hex and Hex B enzymatic activities measured in individuals heterozygous for deltaTG and a null allele, IVS-2 + 1G-->A (G-->A), were approximately 30% lower than the activities of G-->A/WT individuals. Analysis of the HEXB mRNA from leukocytes of deltaTG/WT individuals by RT-PCR of the 3'UTR showed that the deltaTG allele is present at lower level than the WT allele. By polyacrylamide gel electrophoresis, it was determined that a PCR fragment containing the +TG version of the 3'UTR of the HEXB gene had an irregular structure. On inspection of genes containing a TG dinucleotide upstream from the polyadenylation signal we found that this dinucleotide was part of a conserved sequence (TGTTTT) immersed in a A/T-rich region. This sequence arrangement was present in more than 40% analyzed eukaryotic mRNAs, including in the human, mouse and cat HEXB genes. The significance of the TG deletion in reference to Sandhoff disease as well as the possible functional role of the consensus sequence and the DNA structure of the 3'UTR are considered.

3' Untranslated Regions↗