Biomedical subjects

R M Tanguay

Publications and source records attributed to R M Tanguay.

At least 19 recordsLinked to original sources

PubMed2001

Perinatal expression of heat shock proteins HSC 70 and HSP 70 in neural and non neural tissues of the piglet.

Stress of different kinds during early perinatal life can result in severe consequences for further development. To determine possible involvement of heat shock proteins in brain development, the expression of HSC 70 and HSP 70 was determined in brain regions (cerebellum, cortex, hippocampus, hypothalamus and striatum) and non neural tissues (liver, lungs and kidneys) at birth and during early development of the piglet. In brain regions, HSC 70 expression was decreased during the few hours following birth. With the exception of cortex, hippocampus and kidney where a decrease of expression was observed, HSP 70 did not show significant changes during early development. These results are discussed in terms of using the piglet model of development to study the effect of different kinds of stress like hypoxia or temperature changes on brain development.

Animals↗

PubMed2001

A missense mutation (Q279R) in the fumarylacetoacetate hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation.

BACKGROUND: Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules. RESULTS: Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. CONCLUSION: These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.

DNA Mutational Analysis↗

PubMed2001

Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.

Animals↗

PubMed2000

The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization.

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1-17 and an unknown number of residues between 17-28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8-13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 of Drosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.

Amino Acids↗

PubMed2000

Characterization of the heat-shock protein 60 chaperonin from Onchocerca volvulus.

Chaperonin 60 (cpn60) belongs to the group of ubiquitous molecular chaperones that comprise the heat shock proteins, nucleoplasmins and chaperonins. Antibodies to recombinant CPN60 from humans was used to screen a cDNA library of Onchocerca volvulus and antigen-positive clones were selected. Sequencing of the DNA inserts confirmed their identity as cpn60 transcripts. These are distinct from a cpn60 sequence recorded previously from O. volvulus (GenBank accession number Y09416) that appears to be of endobacterial origin, rather than derived from the parasite itself. The full-length sequence of the cDNA (designated Ov-cpn60) codes for a protein of 64.3kDa (598 amino acid residues) and shares significant identity with homologous gene products from Caenorhabditis elegans (72%), humans (69%), yeast (53%) and Escherichia coli (50%). The endobacterial and parasite sequences are 41% conserved. Ov-CPN60 migrates with an apparent molecular mass of 65kDa on SDS-PAGE and is present in all life-cycle stages, as determined by immunoblotting with rabbit antibodies raised against the recombinant protein. Immunogold electron microscopy identified the protein within mitochondria, as expected, but also in extra-mitochondrial sites, including inclusion bodies of the glandular oesophagus (in infective larvae), the uterine wall, cytosol of developing spermatids, and the hypodermis and cuticle. Endobacteria were also labelled, indicating cross-reactivity between CPN60 from the parasite and its intracellular symbiont. In human infections, serum antibodies to Ov-CPN60 were present in only 11% of cases from Ecuador, but in 81-89% of subjects in three separate foci from West Africa. There was no relationship between antibody levels and age, sex, or infection intensity, and no consistent association between the serological response and immune status. An evaluation of antibody specificities in individual sera revealed a mixture of parasite-specific and host crossreactive anti-CPN60 antibodies, the ratio of which varied amongst geographic areas. It is concluded that antibody responses to Ov-CPN60 are unlikely to contribute either to host protection or pathology in onchocerciasis.

Amino Acid Sequence↗

PubMed2000

Hepatocellular carcinoma despite long-term survival in chronic tyrosinaemia I.

Tyrosinaemia I (fumarylacetoacetate hydrolase deficiency) is an autosomal recessive inborn error of tyrosine metabolism that produces liver failure in infancy or a more chronic course of liver disease with cirrhosis, often complicated by hepatocellular carcinoma, in childhood or early adolescence. We studied a 37-year-old woman with tyrosinaemia I whose severe liver disease in infancy and rickets during childhood resolved with dietary therapy. From 14 years of age she resumed an unrestricted diet with the continued presence of the biochemical features of tyrosinaemia, yet maintained normal liver function. In adult years she accumulated only small amounts of succinylacetone. Despite this evolution to a mild biochemical and clinical phenotype, she eventually developed hepatocellular carcinoma. Her fumarylacetoacetate hydrolase genotype consists of a splice mutation, IVS6-1g>t, and a novel missense mutation, Q279R. Studies of resected liver demonstrated the absence of hydrolytic activity and of immunological expression of fumarylacetoacetate hydrolase in liver tumour. In nontumoral areas, however, 53% of normal hydrolytic activity and immunologically present fumarylacetoacetate hydrolase was found. This case demonstrates the high risk of liver cancer in tyrosinaemia I even in a seemingly favourable biological environment.

Adult↗

PubMed1999

Genotyping of a case of tyrosinaemia type I with normal level of succinylacetone in amniotic fluid.

Tyrosinaemia type I is caused by a deficiency of fumarylacetoacetate hydrolase and mainly affects the liver. This disease is characterized by the presence of a high level of succinylacetone. This metabolite has been used for prenatal diagnosis from amniotic fluid samples. One case with a normal level of succinylacetone in amniotic fluid has recently been described (Grenier et al., 1996). Here, we report that this patient is a compound heterozygote for two known mutations: E364X and IVS6-1g-->t. The low level of succinylacetone cannot be explained by these mutations.

Amino Acid Metabolism, Inborn Errors↗

PubMed1999

Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.

Hereditary tyrosinemia type I is the most severe metabolic disease of the tyrosine catabolic pathway mainly affecting the liver. It is caused by deficiency of fumarylacetoacetate hydrolase, which prevents degradation of the toxic metabolite fumarylacetoacetate (FAA). We report here that FAA induces common effects (i.e., cell cycle arrest and apoptosis) in both human (HepG2) and rodent (Chinese hamster V79) cells, effects that seem to be temporally related. Both the antiproliferative and apoptosis-inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L-buthionine-(S,R)-sulfoximine (BSO). Short treatment (2 h) with 35 microM FAA/+BSO or 100 microM FAA/-BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post-treatment. In cells treated with 100 microM FAA/-BSO, an inactivation, followed by a rapid over-induction of cyclin B-dependent kinase occurred, which peaked 24 h post-treatment. Maximum levels of caspase-1 and caspase-3 activation were detected at 3 h and 32 h, respectively, whereas release of mitochondrial cytochrome c was maximal at 24-32 h post-treatment. The G2/M peak declined 24 h later, concomitantly with the appearance of a sub-G1, apoptotic population showing typical nucleosomal-sized DNA fragmentation and reduced mitochondrial transmembrane potential (Deltapsi(m)). These events were prevented by the general caspase inhibitor z-VAD-fmk, whereas G2/M arrest and subsequent apoptosis were abolished by GSH-monoethylester or N-acetylcysteine. Other tyrosine metabolites, maleylacetoacetate and succinylacetone, had no antiproliferative effects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA-induced cell cycle disturbance and apoptosis where activation of cyclin B-dependent kinase and caspase-1 are early events preceding mitochondrial cytochrome c release, caspase-3 activation, and Deltapsi(m) loss. -Jorquera, R., Tanguay, R. M. Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.

Acetoacetates↗

PubMed1999

The nuclear chronicles: gene transcription and molecular traveling.

The transfer and processing of an RNA transcript from its locus of transcription on chromatin through the nuclear membrane to its site of translation on cytoplasmic ribosomes is a long and complex journey involving numerous processes and interactions with various macromolecules. These various steps that regulate gene expression were the subject of the 9th Winternational Symposium of the Canadian Society of Biochemistry and Molecular & Cell Biology held at Manoir du Lac Delage, a small resort centre north of Quebec City on February 12-15, 1999.

Animals↗

PubMed1999

SUMO/sentrin: protein modifiers regulating important cellular functions.

Regulation of protein functions can be achieved by posttranslational protein modifications. One of the most studied modifications has been conjugation to ubiquitin, which mainly targets substrate proteins for degradation by the 26 S proteasome. Recently, SUMO/sentrin, a ubiquitin-like protein has been characterized. This evolutionary conserved protein is conjugated to specific proteins in a way similar, but not identical, to ubiquitin and seems also to be involved in the regulation of protein localization or function. An increasing number of SUMO/sentrin substrates are currently described. We focus here on three major substrates of modification by SUMO: RanGAP1, PML, and IkappaBalpha proteins. These different examples illustrate how SUMO conjugation may be involved in the control of the level of critical proteins within the cell or in the modulation of subcellular localization and nucleocytoplasmic trafficking.

Animals↗

PubMed1998

Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small heat shock proteins in Drosophila melanogaster.

In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23. Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27. In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns. The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9. Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S. cerevisiae ubc9ts mutant. Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27. The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development. The significance of the sHsp-Ubc9 interaction is discussed.

Amino Acid Sequence↗

PubMed1998

Different clinical forms of hereditary tyrosinemia (type I) in patients with identical genotypes.

Hereditary tyrosinemia type I (HTI, McKusick 276700) is an autosomal recessive disease caused by deficient fumarylacetoacetate hydrolase (FAH, EC 3.7.1.2) activity. HTI is characterized by progressive liver dysfunction with nodular cirrhosis often leading to hepatocellular carcinoma. Two extremes of the clinical phenotype have been described: the "acute" (severe, early onset and death) and "chronic" (delayed onset and slow course) phenotype. Allelic heterogeneity and/or mutation reversion in hepatic cells have been proposed earlier to explain the clinical heterogeneity. Two probands (one "acute," one "chronic") from the French-Canadian isolate where HTI is prevalent were studied. Both were homozygous (germ line) for the severe splice mutation IVS12 + 5g --> a; both showed liver mosaicism for FAH immunoreactivity with evidence for mutation reversion to heterozygosity (IVS12 + 5g --> a/+) in FAH-stained nodules as shown by amplification of DNA extracted from microdissected nodules. Western blot analysis of proteins from a reverted FAH-expressing nodule showed 29 +/- 3% FAH immunoreactive material as compared to an average normal liver. This was consistent with the measured FAA hydrolytic activity (25%) in this large regenerating nodule. These findings show that genotypic heterogeneity is not a sufficient explanation for clinical heterogeneity and implicate epigenetic and other factors modifying the phenotype in HTI.

Acute Disease↗

PubMed1998

Expression of heat shock proteins in mouse skin during wound healing.

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Animals↗

PubMed1998

Presence of antibodies to heat stress proteins in workers exposed to benzene and in patients with benzene poisoning.

Heat shock or stress proteins (Hsps) are a group of proteins induced by a large number of xenobiotics, many of which are common in the working and living environment. The biological significance of the presence of antibodies against Hsps in humans is presently unknown. In the present study, 112 workers were selected and divided into four groups on the basis of their level of occupational exposure to benzene: a control group, two groups of workers exposed to either low (< 300 mg/m3) or high concentrations of benzene (> 300 mg/m3) and a group of workers who had experienced benzene poisoning. Blood samples from these workers were assayed for the number of peripheral white blood cells, concentration of hemoglobin, activities of serum superoxide dismutase (SOD), lymphocyte DNA damage and finally for the presence of antibodies to different human heat-shock proteins (Hsp27, Hsp60, Hsp71 and Hsp90). Benzene-poisoned workers showed a high incidence of antibodies against Hsp71 (approximately 40%) which was associated with a decrease in white blood cells (3.84+/-1.13 x 10(9)versus 7.68+/-1.84 x 10(9) in controls) and with an increase in activities of serum SOD (138.43+/-23.15 micro/ml) and lymphocyte DNA damage (18.7%). These data suggest that antibodies against Hsps can potentially be useful biomonitors to assess if workers are experiencing or have experienced abnormal xenobiotic-induced stress within their living and working environment.

Adult↗

PubMed1997

Specific intranucleolar distribution of Hsp70 during heat shock in polytene cells.

Hsp70, the most abundant and conserved heat shock protein, has been described as strongly concentrating in the nucleolus during heat shock. The important metabolic processes that take place in the nucleolus, rDNA transcription, processing, and assembling with ribosomal proteins, and the nucleolar architecture itself are very sensitive to temperature changes. In this work, we have analyzed in detail the nucleolar changes, in structure and activity, induced by temperature in Chironomus thummi salivary gland cells and the fine subnucleolar localization of Hsp70 during heat shock. The optimum temperature chosen to induce the heat shock response was 35 degrees C. Under these conditions transcription of heat shock genes, inactivation of previously active genes and maximum synthesis of Hsps take place, while survival of larvae and recovery were ensured. After 1 h at 35 degrees C, nucleoli change from a uniform control pattern to a segregated pattern of nucleolar components that can be observed even at the light microscopic level. The dense fibrillar component (DFC) and the granular component appeared perfectly differentiated and spatially separated, the former occupying mainly the central inner region surrounded by a rim of granular component. Hsp70 was specifically localized within the DFC upon heat shock as shown by immunolocalization by both light and electron microscopy. Pulse labeling with [3H]uridine proves that rRNA transcription continues during heat shock. The pattern of Hsp70 distribution within the nucleolus correlates with that of newly produced rRNA transcripts. Hsp70 also colocalizes with RNA polymerase I, both being restricted to the DFC. These data show that the DFC seems to be the intranucleolar target for Hsp70 in heat-shocked cells. We discuss these results in relation to the possible function of Hsp70 in the first steps of preribosome synthesis.

Animals↗

PubMed1997

The mutagenicity of the tyrosine metabolite, fumarylacetoacetate, is enhanced by glutathione depletion.

The toxicity of tyrosine metabolites has been suggested, but not proven, to play a role in the ethiopathogenesis of hepatic alterations observed in hereditary tyrosinemia type I (HT I), a metabolic disease caused by a deficiency of the last enzyme in the tyrosine catabolic pathway, fumarylacetoacetate hydrolase. One of these metabolites, fumarylacetoacetate (FAA), is mutagenic in Chinese hamster V79 cells. We report here that FAA is a powerful glutathione depletor in this cell system. Moreover, the mutagenicity of FAA (100 microM) is potentiated by depletion of cellular glutathione (12% of control levels) by pretreatment with L-buthionine-(S,R)-sulphoximine. In this case, the mutation frequency induced by FAA is 10 times higher than in untreated, control cells. This enhancement is abolished by a partial replenishment of intracellular glutathione (32% of control levels) prior to FAA treatment. Reactive oxygen species are not generated during FAA treatment of glutathione-depleted or undepleted cells. Although the mechanism(s) underlying the mutagenic activity of FAA remains to be identified, these results show that the glutathione depletion activity of FAA may play an important role in the manifestation of its mutagenicity which likely contributes to the HT I-associated liver pathologies.

Acetoacetates↗

PubMed1997

Mutations in the fumarylacetoacetate hydrolase gene causing hereditary tyrosinemia type I: overview.

Tyrosinemia type I is an inborn error of metabolism caused by a deficiency in the last enzyme of the tyrosine catabolic pathway, fumarylacetoacetate hydrolase (FAH). The disease has been reported worldwide with varying incidence. Recently, there has been considerable progress in identifying mutations in the FAH gene. At present 26 mutations have been reported, all consisting of single base substitutions resulting in 16 amino acid replacements, one silent mutation causing a splicing defect, five nonsense codons, and four putative splicing defects. The location of these mutations is spread over the entire FAH gene, with a particular clustering between amino acid residues 230 and 250. The identification of these mutations in subpopulations and groups at high risk should help in the diagnosis of, and genetic counseling for, HT1. We describe all these 26 mutations reported so far and their implication in diagnosis and carrier detection.

Amino Acid Metabolism, Inborn Errors↗

PubMed1997

Preliminary studies on the relationship between autoantibodies to heat stress proteins and heat injury of pilots during acute heat stress.

Comparison in the heart rate, oral temperature and lymphocyte DNA damage during heat stress was made in pilots with negative antibodies to heat stress proteins (HSPs) and those with positive antibodies in the man-made climate room with Western blot and comet assay. Our results showed that the increase in oral temperature, heart rate and lymphocyte DNA damage in pilots with the positive antibodies to HSPs were higher than those in pilots with the negative antibodies during heat stress. These results indicated that the presence of autoantibodies in plasma of pilots might reflect heat damage and high sensitivity to heat.

Adult↗