Search

Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by approximately 75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent.

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.

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.

Hypertyrosinemia encompasses several entities, of which tyrosinemia type I (or hepatorenal tyrosinemia, HT1) results in the most extensive clinical and pathological manifestations involving mainly the liver, kidney, and peripheral nerves. The clinical findings range from a severe hepatopathy of early infancy to chronic liver disease and rickets in the older child; gradual refinements in the diagnosis and medical management of this disorder have greatly altered its natural course, mirroring recent advances in the field of metabolic diseases in the past quarter century. Hepatorenal tyrosinemia is the inborn error with the highest incidence of progression to hepatocellular carcinoma, likely due to profound mutagenic effects and influences on the cell cycle by accumulated metabolites. The appropriate follow-up of patients with cirrhosis, the proper timing of liver transplantation in the prevention of carcinoma, and the long-term evolution of chronic renal disease remain important unresolved issues. The introduction of a new pharmacologic agent, NTBC, holds the hope of significantly alleviating some of the burdens of this disease. Mouse models of this disease have permitted the exploration of newer treatment modalities, such as gene therapy by viral vectors, including ex vivo and in utero methods. Finally, recent observations on spontaneous genetic reversion of the mutation in HT1 livers challenge conventional concepts in human genetics.

Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.

Antibodies against heat shock or stress proteins (Hsps) have been reported in a number of diseases in which they may be involved in the pathogenesis of the disease or may be of use for prognosis. Heat-induced diseases, such as heat cramps, heat exhaustion, or heat stroke, are frequent in hot working or living environments. There are still few investigations on the presence and possible significance of autoantibodies against Hsps in heat-induced illnesses. Using an immunoblotting technique with recombinant human Hsps, we analyzed the presence and titers of antibodies against Hsp60, Hsp71, and Hsp90alpha, and Hsp90beta in a group of 42 young male patients who presented with acute heat-induced illness during training. We also examined the presence of antibody against Hsp71 in a second group of 57 patients with acute heat-induced illness and measured the changes in titers of anti-Hsp71 antibodies in 9 patients hospitalized by emergency physicians. In the first group of young persons exercising in a hot environment, the occurrence of antibodies against Hsp71 and Hsp90alpha was significantly higher among individuals with symptoms of heat-induced illness (P < 0.05) than in the matched group of nonaffected exercising individuals. Moreover titers of antibody against Hsp71 were higher in individuals of the severe and mild heat-induced illness groups, the highest titer being found in the most severe cases. The results from the second group of 57 heat-affected patients exposed to extreme heat were similar. Again, patients with the more severe heat-induced symptoms showed a significantly higher incidence of antibodies to Hsp71 than controls and the titer of anti-Hsp71 was higher in the severely affected group. Finally, in a study of 9 patients, it was observed that the titer of anti-Hsp71 decreased during recovery from severe heat symptoms. These results suggest that measurement of antibodies to Hsps may be useful in assessing how individuals are responding to abnormal stress within their living and working environment and may be used as one biomarker to evaluate their susceptibility to heat-induced diseases.

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

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.

The internal light-regulatory element (iLRE) of ferredoxin (Fed-1) mRNA, comprising the 5' leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translation-dependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5' leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.

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.

Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2alpha and rtAhR2beta), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2alpha, zebrafish AhR2, and human AhR), while rainbow trout AhR2beta had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2alpha was significantly more active than AhR2beta. However, rainbow trout AhR2beta is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2beta may have evolved to serve a different physiological function than AhR2alpha in salmonid fish species., (Copyright 1999 Academic Press.)

Objective: To study whether physiological and psychological stresses during parachuting jumps may result in biochemical changes of plasma in parachutists., Method: Differences in the levels of hormones (cortisol, growth hormone, insulin, pancreatic glucagon, endothelin, angiotonin I and II, aldosterone), activities of enzymes (superoxide dismutase, glutathione peroxidase, glutathione S transferase), levels of the free radical damage indicator malondialdehyde (MDA), tumor necrosis factor alpha (TNF-alpha), and the main heat stress protein, HSP70, in the plasma and serum were investigated in control (non-parachuting) and parachuting paratroops., Result: Significantly higher levels of serum hormones such as growth hormone, insulin, angiotonin 1, renin activities, as well as MDA and plasma TNF-alpha and HSP70 were observed in the parachuting group., Conclusion: Whether these changes can potentially serve as useful biomarkers to assess possible abnormal stress in parachutists and to evaluate the health condition and to select parachutists remains to be further studied.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.

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.

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.

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.

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., (Copyright 1998 Academic Press.)

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.

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.

Mice lacking the enzyme fumarylacetoacetate hydrolase (FAH) have symptoms similar to humans with the disease hereditary tyrosinemia type I (HT1). FAH-deficient mice were injected with a first-generation adenoviral vector expressing the human FAH gene and followed for up to 9 months. Nontreated FAH mutant control mice died within 6 weeks from fulminant liver failure, whereas FAH adenovirus-infected animals survived until sacrifice at 2-9 months. Nine of 13 virus-treated animals developed hepatocellular cancer. Immunohistochemical analysis revealed a mosaic of FAH-deficient and FAH-positive cells in all animals and liver function tests were improved compared to controls. Even mice harvested 9 months after viral infection had > 50% FAH-positive cells. These results demonstrate the strong selective advantage of FAH-expressing cells in an FAH-deficient liver but also illustrate the danger of carcinomas arising from FAH-deficient hepatocytes in HT1.

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.

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.

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.

The putative role of the dopaminergic system in sleep bruxism (SB) was studied in a double-blind clinical trial by using low doses of short-term L-dopa in combination with benserazide. We recorded 10 patients with SB in our sleep laboratory for 3 consecutive nights. The first night was for habituation to the laboratory environment. During the second and the third nights, the patients received two doses of either L-dopa or a placebo in a crossover fashion: the first dose 1 h before bedtime and the second, 4 h after the first one. The order of administration was reversed in half the patients. The efficacy of L-dopa was analyzed by using multilevel models. L-Dopa resulted in a significant decrease in the average number of bruxism episodes per hour of sleep, as well as in a significant reduction in the average value of the root-mean-square (RMS) electromyography (EMG) level per bruxism burst. This indicates that L-dopa exerts an attenuating effect on SB. In addition, L-dopa caused a reduction in the variance in RMS values, which suggests that L-dopa normalizes the EMG activity patterns associated with SB.

Induction of heat shock proteins (Hsps), Hsp72 in particular, has been associated with myocardial protection. Since a decreased Hsp response has been reported to occur with aging, it was of interest to determine if hearts from aged animals also demonstrate an altered heat shock response and subsequent myocardial protection. Adult (6 months old) and aged (22 months old) Fischer 344 rats were heat stressed by raising their rectal temperatures to 41 degrees C for 10 min. At selected times following heat stress (0-24 h) hearts were examined for heat shock transcription factor trimerization and DNA-binding activity (Hsf1 activation), Hsp72 mRNA accumulation, Hsp72 and Hsf1 protein content, as well as, protection from ischemia using the Langendorff isolated heart model. Following heat stress, hearts from aged animals demonstrated a 47% reduction in Hsf1 activation, a reduction in Hsp72 mRNA and a 35% reduction in Hsp72 protein content, compared to hearts from adults. Interestingly, myocardial Hsf1 protein content was similar between aged and adult animals. Hearts from heat stressed adult animals (24-h prior) demonstrated an enhanced postischemic recovery as indicated by a greater recovery of left ventricular pressure and rate of contraction (P < 0.05), while hearts from heat stressed aged animals failed to demonstrate an enhanced postischemic recovery. These results suggest that hearts from aged animals exhibit an impaired ability to produce the protective Hsps and thus, may explain, at least in part, the increased susceptibility of aged hearts to stress.

Antibodies to the ubiquitous group of stress proteins known as heat shock proteins (Hsps) have been found to be associated with a number of diseases in humans. Hsps are known to be induced by certain xenobiotics, some of which are common in the working environment. The biological significance of the presence of such autoantibodies is presently unclear. In the present study, we used immunoblotting to investigate the presence of antibodies against the different stress proteins, Hsp27, Hsp60, Hsp71, Hsc (heat shock cognate) 73 and Hsp89 alpha and beta in groups of workers exposed to high temperature or carbon monoxide. These data were related to a detailed clinical evaluation and to various laboratory measurements including electrocardiogram (ECG), B echogram, white blood cell counts and typing, the activity of alanine aminotransferase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) and lymphocyte DNA damage. Antibodies to Hsp27 and Hsp71 were found more frequently in the high temperature and carbon monoxide-exposed groups than in controls (P < 0.05). The carbon monoxide-exposed group showed the highest incidence of anti-Hsp antibodies. Anti-Hsp60 antibodies were only detected in workers exposed to high temperature or carbon monoxide. The percentage of workers with abnormal ECG, B echogram changes and displaying hepatitis B antigen (HBsAg) was higher in the carbon monoxide group than in the control group (P < 0.05). There was a significant increase in the activity of ALT in the high temperature and carbon monoxide groups and in the activities of ACP and ALP in the carbon monoxide group (P < 0.05). The extent of DNA damage measured in lymphocytes was higher in workers from the high temperature and carbon monoxide-exposed groups. We suggest that the increased frequency of antibodies to Hsps is the result of these damages of the release of denatured Hsps and of a decrease in the phagocytic ability of macrophages in these workers. The data gathered in the present study show a statistical relation between the occurrence of antibodies against Hsps and the frequency of health problems in workers and suggest a potential role for the antibodies as useful biomarkers to assess whether workers are experiencing environmental stress.

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42 degrees C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

The effect of increasing the length of the 3'-untranslated region (UTR) on expression of luciferase mRNA was examined in transiently transfected carrot protoplasts. The effect of the 3'-UTR on both poly(A)- and poly(A)+ mRNA was examined. A nested set of constructs in which the 3'-UTR increased from 4 to 104 bases was generated by the introduction and reiteration of a 20 base sequence downstream of the luc stop codon. For poly(A)- mRNA, there was a consistent increase in expression when the length of the 3'-UTR was increased from 4 to 104 bases. For poly(A)+ mRNA, expression increased 18-fold when the length of the 3'-UTR was increased from 7 to 27 bases. Further increases in the length of the 3'-UTR did not affect expression. The increase in expression was largely due to an increase in translational efficiency. These data suggest that the length of the 3'-UTR plays an important role in determining the extent to which a poly(A) tail can stimulate the translation of an mRNA.

Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5' region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of the rat hindlimb which differ in the constitutive expression of Hsp70, were assessed for HSF activation following heat shock. Mobility shift gel analyses demonstrated that HSF activation was detectable in extracts from all muscles following heat shock regardless of Hsp70 content. However, muscles comprised predominantly of slow/Type I fibers (soleus) demonstrated a greater HSF activation, as well as a faster HSF activation and inactivation, than muscles comprised predominantly of fast/Type II fibers (white gastrocnemius). In addition, muscles pretreated by two heat shocks (24 h apart) demonstrated a stronger HSF activation than muscles subjected to only one heat shock. Thus, results from cell culture models demonstrating that tissue levels of Hsp70 are inversely correlated with HSF activation, may not apply to the muscles of an intact animal.

The expression of two small heat shock proteins (sHsp), Hsp23 and Hsp27, was examined by immunological approaches in the eye of Drosophila melanogaster. Neither Hsp23 nor Hsp27 is detectable in unstressed (23 degrees C) eyes but both proteins are induced by heat shock (35 degrees C). In response to heat stress, Hsp27 is expressed in all cells of the ommatidium including the cone, pigment and photoreceptor cells. However, the heat-induced expression of Hsp23 is restricted to a single cell type of the ommatidium, the cone cells, suggesting that Hsp23 is regulated by specific mechanisms acting to inhibit the expression of this polypeptide in some ommatidial cells. The cell-specific induction of Hsp23 under stress conditions does not seem to be regulated by the Drosophila melanogaster heat shock transcriptional factor (DmHSF). In both unstressed and stressed conditions, DmHSF is detected in all the different types of ommatidial cells where it is found associated with the nucleus. These observations suggest that factors, other than the heat shock transcriptional factor, are involved in regulating the expression of the hsp23 gene under stress conditions.

Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

Stress-induced regulation of the 72 kD heat shock protein (HSP 72), the major stress inducible protein in mammalian cells, is mediated by the activation and binding of a heat shock transcription factor (HSF) to a specific sequence in the 5' region of the promoter termed the heat shock element (HSE). In agreement with this regulation, HSP 72 is absent in most cells under unstressed conditions but is rapidly synthesized following exposure to protein damaging stressors. An exception is the skeletal muscle, where HSP 72 is constitutively expressed in muscles that express the beta myosin heavy chain (beta-MHC) protein. Since beta-MHC is also expressed in the ventricles of large mammals, we have examined if HSP 72 was also constitutively expressed in beta-MHC positive hearts. Chambers of the heart muscle from Yorkshire swine were examined for alpha-MHC, beta-MHC and HSP 72 content. HSF:HSE activation was also assessed by gel shift analyses. In the swine heart, atria and ventricles differed in their alpha-MHC and beta-MHC protein content but all expressed a high HSP 72 content. Gel shift analyses demonstrated no HSF:HSE binding in extracts from unstressed swine hearts. These results indicate that HSP 72 is constitutively expressed in all portions of the swine heart and this expression may not be dependent on an HSF:HSE interaction.

Drosophila sHSPs (small heat shock proteins) are expressed in the absence of stress in specific regions of the central nervous system and in gonads of young adults flies. In these two organs, the sHSPs show a cell-specific and developmental stage-specific pattern of expression suggesting distinct regulation and function(s) of each individual sHSP (R. Marin et al., Dev. Genet. 14, 69-77, 1993). Since mammalian HSP27 has been reported to be phosphorylated through a complex novel cascade implicating distinct kinases, we examined whether two of the sHSPs (HSP27 and HSP23) exist in different isoforms as a result of posttranslational modification in vivo. HSP27 and HSP23 were analyzed in various tissues in unstressed and heat-shocked flies. Four isoforms of HSP27 were found to be constitutively expressed in the nervous system and in testes and two in ovaries. The proportion of these isoforms relative to each other was specific to a given tissue. In the case of HSP23, two isoforms were expressed in the heads and in testes of unstressed flies. In ovaries, a low level of a single isoform of HSP23 was found. Heat shock caused an increase in the amount of preexisting HSP27 and HSP23 and the appearance of additional isoforms in ovaries. Susceptibility to phosphatase treatment indicated that isoforms of HSP27 were phosphoproteins. This was further supported by in vitro experiments in which Drosophila sHSPs were incubated with purified Chinese hamster HSP27 kinase. Only HSP27 was shown to be a substrate of this mammalian HSP27 kinase. The present data suggest that tissue- and HSP-specific posttranslational modification systems may modulate the function of these proteins in different cell types. Furthermore, the signal transduction pathways leading to phosphorylation of the sHSPs are conserved between mammals and Drosophila, and the sHSP kinase cascade may be developmentally regulated.

Hereditary tyrosinaemia type I (HTI), an autosomal recessive inborn error of metabolism, is caused by a deficiency of the enzyme fumarylacetoacetate hydrolase. The highest incidence of HTI is observed in the Saguenay-Lac-St-Jean region (SLSJ) (Québec, Canada), where 1 out of 22 individuals is thought to be a carrier. A splice mutation (IVS12 + 5G-->A) has recently been identified in this particular region. Here, we have determined the frequency of this mutation in a population of obligate carriers from the SLSJ region by allele-specific oligonucleotide hybridization and a method using a restriction enzyme digestion. Over 95 per cent of the HTI carriers were found to have the IVS12 + 5G-->A splice mutation. Screening for this mutation based on the two methods reported here is thus a reliable and rapid way of detecting carriers of hereditary tyrosinaemia type I in that region at high risk.

Direct counting of axon terminals (varicosities) labeled by uptake/storage of a tritiated monoamine provides a means to test radioligands of the corresponding membrane transporter as quantitative markers of regional monoamine innervation density in brain tissue. In autoradiographs from alternate rat brain slices, counts of [3H]5-HT-labeled axon terminals were matched with densitometric measurements of the specific binding of tritiated cyanoimipramine (CYI), citalopram (CITAL), and 6-nitroquipazine (6-NTQ), under conditions of hypo-, normo-, or hyper-5-HT innervation of the neostriatum. A total of 267 pairs of data were subjected to a multilevel analysis (iterative generalized least square procedure). With all three ligands, there was a linear relationship between the density of 5-HT innervation and the density of specific binding and no change in the slope of the regression lines as a function of 5-HT innervation density. Thus, none of these ligands gave any sign of down- or up-regulation of the 5-HT transporter consequent to 5-HT hypo- or hyper-innervation. The regression lines for CYI and CITAL were not significantly different from one another and crossed the ordinate near zero, whereas the regression line for 6-NTQ was less steep and had a higher intercept with the ordinate. In addition, the dispersion of values around the regression line (residuals) was lower with CYI and CITAL than 6-NTQ. It was concluded that both CYI and CITAL may serve as quantitative markers of 5-HT innervation density, at least in vitro, whereas 6-NTQ demonstrates a certain lack of specificity and sensitivity. Further work will be needed to assess the potential of CYI and CITAL for positron emission tomographic studies of living brain. Such empirical testing should also be applicable for screening radioligands of the dopamine or the noradrenaline transporters.

Ssb1p and ssb2p are two members of the hsp70 family in yeast. Up to now there has been no evidence to indicate any differences between these two members of the hsp70 family, and it was suggested that ssb1p and ssb2p were 99% identical. Here we show that an antibody prepared against the C-terminal domain of human hsp71 recognizes specifically ssb1p out of the eight hsp(c)70s in Saccharomyces cerevisiae. An amino acid peptide sequence at the C-terminal end (VTATDKSTGK) is suggested to be the sequence which has high homology between ssb1p and hu-hsp71 and to be responsible for the specificity of recognition of this unique member of the hsp70 family. Using this antibody in immunoblot assays, we have determined the cellular content of ssb1p after heat shock and at different growth temperatures. Ssb1p is shown to be degraded during heat shock treatment while it shows a higher level of expression at low temperatures.

Type I hereditary tyrosinemia results from an inherited deficiency in fumarylacetoacetate hydrolase, the enzyme involved in the last step in tyrosine catabolic pathway. The cloning of the cDNA encoding FAH in human has opened the way to genetic treatment of HT 1. We have constructed recombinant retroviral vectors carrying the cDNA encoding human FAH. In the present report we show that these vectors are able to restore FAH activity stably in primary fibroblasts from HT 1 patients and at high level. The possibility to express FAH stably in deficient patients represents a first step towards future gene therapy for type I hereditary tyrosinemia and may help to decipher the pathogenesis of the disease at the molecular level.

In this study, we have examined the effects of exposure to high temperature, carbon monoxide or a combination of both conditions in a model system, the rat and in industrial workers. In the rat liver, HSP70 mRNA and HSP70 synthesis were measured by dot hybridization and western blot. The results showed that after a heat stress HSP70 mRNA and its product, HSP70 increased significantly and there was a synergism in the combined effects of high temperature and carbon monoxide exposure on the induction of HSP70 mRNA and HSP70 synthesis. Heat played a major role in this induction. The presence of antibodies to human HSP27, HSP60, HSP70, HSC73, HSP89 alpha and beta in workers exposed to heat, carbon monoxide was also measured by western blot using purified HSPs as antigens. Plasma free amino acids were measured in the same group of workers. The incidence of antibodies to HSP27 and HSP70 was significantly higher in the workers working in an environment with extreme heat, and high carbon monoxide emission than in a control group. The carbon monoxide exposed group showed the highest incidence of antibodies to HSPs. Although our previous results indicated that workers had an insufficient protein intake, plasma free amino acids tended to increase, especially in methionine and tryptophan two kinds of amino acids which are absent from the main stress protein, HSP70. These results suggest that the major problems that these workers may face are how to facilitate the use of plasma free amino acids and reduce the inhibition of synthesis of normal proteins when they are exposed to occupational harmful factors. These results also add new information on the measurement of HSPs as a potential biomonitor to assess whether organisms are experiencing metabolic stress within their environment.

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.

In the past, fixed prostheses were believed to be more efficient implant-supported devices than removable types for edentulous patients. However, this hypothesis was never properly tested. Therefore, a within-subject crossover clinical trial was designed in which 145 completely edentulous subjects were tested wearing implant-supported mandibular fixed prostheses and long-bar overdentures. Eight subjects received the fixed appliance first and seven the removable type. The patients' perceptions of various characteristics of the implant-supported prostheses were measured after a minimum of two months' adaptation. Mandibular movements and jaw muscle electromyographic activity were recorded while the patients chewed five standard-sized test foods: bread, apple, hard cheese, sausage, and raw carrot. The prostheses were then changed, worn for the same period of adaptation, and the procedures repeated. There were three test sessions per prosthesis, and each included five trials per food. The measurements were repeated three times at one-week intervals. Mastication time was found to be shorter for three foods (bread, cheese, and sausage) when subjects wore the long-bar overdenture. The vertical amplitude of the masticatory strokes was significantly less with the overdenture for all foods except carrot. Cycle duration was significantly longer with the overdenture for sausage and carrot. Contrary to what might be expected, the long-bar overdenture appears to be no less efficient than the fixed prosthesis. Furthermore, these data suggest that patients are capable of adapting their masticatory movements to the characteristics of the two prostheses.

Background: Hereditary tyrosinemia type I is an autosomal recessive inborn error of metabolism caused by a deficiency of the enzyme fumarylacetoacetate hydrolase. The disorder clusters in the Saguenay-Lac-St.-Jean area of Quebec. In this region, 1 of 1846 newborns is affected and 1 of every 22 persons is thought to be a carrier. Recently, we identified a splice mutation and two nonsense mutations in the fumarylacetoacetate hydrolase gene in two patients from Quebec with tyrosinemia type I., Methods: We used allele-specific-oligonucleotide hybridization to examine the frequency of these three candidate mutations in patients with tyrosinemia type I and in the population of Quebec., Results: The splice mutation was found in 100 percent of patients from the Saguenay-Lac-St.-Jean area and in 28 percent of patients from other regions of the world. Of 25 patients from the Saguenay-Lac-St.-Jean region, 20 (80 percent) were homozygous for this mutation, a guanine-to-adenine change in the splice-donor sequence in intron 12 of the gene, indicating that it causes most cases of tyrosinemia type I in the region. The frequency of carrier status, based on screening of blood spots from newborns, was about 1 per 25 in the Saguenay-Lac-St.-Jean population and about 1 per 66 overall in Quebec., Conclusions: This study identified the most prevalent mutation causing hereditary tyrosinemia in French Canada; it also showed the feasibility of DNA-based testing for carriers in the population at risk.

Hereditary tyrosinemia type 1 (HT1), a severe inborn error of tyrosine catabolism, is caused by deficiency of the terminal enzyme, fumarylacetoacetate hydrolase (FAH). The highest reported frequency of HT1 is in the French Canadian population, especially in the Saguenay-Lac-St-Jean region. Using human FAH cDNA probes, we have identified 10 haplotypes with TaqI, KpnI, RsaI, BglII, and MspI RFLPs in 118 normal chromosomes from the French Canadian population. Interestingly, in 29 HT1 children, a prevalent haplotype, haplotype 6, was found to be strongly associated with the disease, at a frequency of 90% of alleles, as compared with approximately 18% in 35 control individuals. This increased to 96% in the 24 patients originating from Saguenay-Lac-St-Jean. These results suggest that one or only a few prevailing mutations are responsible for most of the HT1 cases in Saguenay-Lac-St-Jean. Since most patients were found to be homozygous for a specific haplotype in this population, FAH RFLPs have permitted simple carrier detection in nine different informative HT1 families, with a confidence level of 99.9%. Heterozygosity rate values obtained from 52 carriers indicated that approximately 88% of families at risk from Saguenay-Lac-St-Jean are fully or partially informative. Prenatal diagnosis was also achieved in an American family. Analysis of 24 HT1 patients from nine countries gave a frequency of approximately 52% for haplotype 6, suggesting a relatively high association, worldwide, of HT1 with this haplotype.

Both the 5' cap (m7GpppN) and the 3' poly(A) tail of eukaryotic mRNAs are important regulators of translation efficiency in vivo. Their function, however, is markedly reduced in in vitro translation systems derived from either rabbit reticulocytes or wheat germ. The impact of exogenous poly(A) on cap-dependent translation was examined in vitro. The translation of uncapped mRNA was preferentially repressed in the presence of exogenous poly(A). As a result, translation became increasingly cap-dependent with the increase in exogenous poly(A). The translation in wheat germ lysate was stimulated by the addition of purified eukaryotic initiation factor (eIF)-4B or eIF-4F; however, addition of poly(A) prevented this eIF-4B- or eIF-4F-mediated stimulation. Addition of eIF-4F or eIF-4B, alone, was not sufficient to restore translation in lysate to which poly(A) had been added. Restoration, however, was observed when eIF-4F, eIF-4B, and eIF-4A were added in combination. These data suggest that 1) exogenous poly(A) may bind to and sequester factors required for translation and 2) that capped messages compete with poly(A) more efficiently for these factors than do uncapped mRNAs. Gel shift analysis of purified initiation factors isolated from wheat germ confirmed that eIF-4B and eIF-4F do in fact form complexes with poly(A) in vitro.

Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.

In a within-subject cross-over clinical trial, psychometric and functional measurements were taken while 15 completely edentulous subjects wore mandibular fixed prostheses and long-bar removable implant-supported prostheses. In this paper, the results of a psychometric assessment are presented. Eight subjects first received the fixed bridge and seven the removable type. After having worn a prosthesis for a minimum of two months, subjects responded to psychometric scales that measured their perceptions of various factors associated with prostheses. They also chewed test foods while masticatory activity was recorded. The prostheses were then changed and the procedures repeated. At the end of the study, patients were asked to choose the prosthesis that they wished to keep. Patients assigned significantly higher scores, on visual analogue scales, to both types of implant-supported prostheses than to their original conventional prostheses for all factors tested, including general satisfaction. However, no statistically significant differences between the two implant-supported prostheses were detected except for the difficulty of chewing carrot, apple, and sausage. For these foods, the fixed prostheses were rated higher. Subjects' responses to category scales were consistent with their responses to the visual analogue scales. These results suggest that, although patients find the fixed bridge to be significantly better for chewing harder foods, there is no difference in their general satisfaction with the two types of prostheses.

Hereditary tyrosinemia type I is a metabolic disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH, EC 3.7.1.2), the last enzyme in the catabolic pathway of tyrosine. The molecular basis of FAH deficiency was examined in five Finnish patients suffering from this severe metabolic disease. No immunoreactive FAH nor enzymatic activity were found in their liver. Direct sequencing of the 14 exons of the FAH gene showed a G to A transition, which predicts a change from tryptophan to a stop codon (TGG-->TGA) at position 262 (W262X). Four of the five patients examined were homozygous for the mutation. Allele specific oligonucleotide hybridization showed a predominance of the W262X mutation in Finland (9 of 10 alleles) and the absence of this mutant allele in patients from other parts of the world. The loss of a BsaJI restriction site in those patients may be used for diagnosis.

Mice homozygous for specific deletions around the albino locus on chromosome 7 die within the first few hours of birth. They have a complex phenotype in liver and kidney, which includes multiple changes in gene expression and ultrastructural abnormalities. On the basis of this phenotype, it was proposed that these deletions remove a regulatory locus, alf or hsdr-1. Recently, we and others showed that the gene for fumarylacetoacetate hydrolase (Fah), an enzyme involved in tyrosine catabolism, was disrupted by the lethal albino deletion c14CoS. The finding that the Fah gene in wild-type mice is highly expressed only in cell types that develop a phenotype in mutants, and the fact that Fah deficiency determines the human liver disease hereditary tyrosinemia type 1 (HT1), suggested that disruption of the Fah gene was responsible for the lethal albino phenotype. To test this hypothesis, we have created lines of mice carrying Fah transgenes. We find that c14CoS homozygotes which express transgenic Fah are complemented for all aspects of the complex lethal albino phenotype. Moreover, the degree to which the phenotype is corrected depends on the level of transgenic Fah expression. These results unequivocally establish Fah as the gene mapping at alf/hsdr-1 and prove that the phenotype depends ultimately on the blockage of tyrosine metabolism. Finally, they suggest lethal albino mice as an animal model for HT1.