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D Leclerc

Publications and source records attributed to D Leclerc.

At least 19 recordsLinked to original sources
PubMed2001

Is the SLC7A10 gene on chromosome 19 a candidate locus for cystinuria?

One of the genes (SLC7A9) that causes cystinuria, an inborn error of amino acid transport, is localized to 19q13. Close examination of human genomic DNA sequences has identified a similar gene (SLC7A10) that also maps to the 19q13.1 region and is highly expressed in kidney. The homologies between SLC7A9 and SLC7A10 are likely the result of gene duplication. SLC7A10 is known to encode a protein with a function similar to that of the SLC7A9 gene product. To determine if mutations in the SLC7A10 gene could also cause cystinuria, we characterized the primary genomic structure and sequenced the 11 exons and surrounding sequences from 10 unrelated patients with cystinuria. We identified one missense mutation which may account for cystinuria in one family. We also observed one intronic change, as well as one silent mutation, that were seen only in cystinuria patients. We therefore suggest that the SLC7A10 gene warrants further investigation as another candidate gene for cystinuria.

Amino Acid Sequence↗
PubMed2001

Changes in the performances of filter media during clogging and cleaning cycles.

The performance of two filter media used in industrial air cleaning were studied both in the initial state (new filter) and after a number of collection and pulse pressure cleaning cycles. The main difference between them is that one has anti-clogging properties and the other does not. The test aerosol is composed of alumina particles with a median volumetric diameter of 2.6 microm (MMAD=4.8 microm) generated at a concentration of 700 mg x m(-3). Filtration took place at a velocity of 2 cm x s(-1). Two parameters, namely pressure drop and efficiency, were monitored according to the collection and cleaning cycles. The comparison of the filtration efficiency of the two media and that of the corresponding industrial dust separator at the end of the cycles showed a close agreement. The separation efficiency calculated with a new medium (corresponding to initial switch-on of the installation) was low and increased very quickly during the cycles. Finally, a phenomenological model was developed to represent the increase in pressure drop of a filter medium after cleaning and was found to be in close agreement with the experimental values.

Equipment Failure Analysis↗
PubMed2001

The product of ORF III in cauliflower mosaic virus interacts with the viral coat protein through its C-terminal proline rich domain.

Using the yeast two-hybrid system, we show that the ORF III product of cauliflower mosaic virus (pIII) interacts through its C-terminus with the viral coat protein. The last five amino acids of pIII were essential for the interaction and virus infectivity. Deletion of the last three amino acids or the mutation F129A decreased the strength of the interaction by 90%. We further show that pIII is closely associated with virus particles found in the inclusion bodies of infected plants but not in viral particles released from the inclusion bodies by urea treatment.

Base Sequence↗
PubMed2001

Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor.

Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.

Amino Acid Sequence↗
PubMed2001

Tetramerization is a conserved feature of the virion-associated protein in plant pararetroviruses.

All plant pararetroviruses belong to the Caulimoviridae family. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus of Caulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.

Amino Acid Sequence↗
PubMed2001

Biochemical characterization of the helper component of Cauliflower mosaic virus.

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Amino Acid Sequence↗
PubMed2000

The effect of high hydrostatic pressure on the strawberry anthocyanins.

Color stability of fruit juice made from strawberries (Fragaria x ananassa, cv. Elsanta) that were subjected to high hydrostatic pressure was studied by measuring the anthocyanin content. High hysrostatic pressure is a method of preservation of food alternative to heat treatment. It is therefore essential to assess the impact of high pressure on color molecules. Samples were pressurized under 200, 400, 600, and 800 MPa for 15 min at a temperature controlled between 18 and 22 degrees C. After application of pressure, the anthocyanin content of the strawberry juice was analyzed by HPLC-UV using a novel isocratic elution system. The high-pressure treated samples were kept at refrigerator temperature (4 degrees C), room temperature (20 degrees C), and 30 degrees C. Two pigments were identified and quantified: pelargonidin 3-glucoside and pelargonidin 3-rutinoside. The highest stability of the anthocyanins was found when strawberries were stored at a temperature of 4 degrees C. High-pressure treatment at 800 MPa led to the lowest losses, at 4 degrees C.

Anthocyanins↗
PubMed1999

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein.

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.

Amino Acid Sequence↗
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

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

Nuclear targeting of the cauliflower mosaic virus coat protein.

The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.

Amino Acid Sequence↗