Metabolic Reprogramming in Amyotrophic Lateral Sclerosis.

Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients' skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients' skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 µmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 µmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 µmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 µmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.

Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Stochastic modeling in toxicokinetics. Application to the in vivo micronucleus assay.

A stochastic model for the in vivo micronucleus assay is presented. This model describes the kinetic of the rate of micronucleated polychromatic erythrocytes induced by the administration of a mutagenic compound. For this, biological assumptions are made both on the erythropoietic system and on the mechanisms of action of the compound. Its pharmacokinetic profile is also taken into account and it is linked to the induced toxicological effect. This model has been evaluated by analyzing the induction of micronuclei is mice bone marrow by a mutagenic compound, 6-mercaptopurine (6-mp). This analysis enabled to make interesting remarks about the induction of micronuclei by 6-mp and to put to light an unsuspected wavy kinetic by optimizing the experimental design of the in vivo micronucleus assay.

Cellular localization of isoprenoid biosynthetic enzymes in Marchantia polymorpha. Uncovering a new role of oil bodies.

Like seed plants, liverworts synthesize and accumulate a myriad of isoprenoid compounds. Using antibodies raised against several isoprenoid biosynthetic enzymes, we investigated their intracellular compartmentation by in situ immunolocalization from Marchantia polymorpha. The enzymes examined were deoxy-xylulose phosphate synthase, geranyl diphosphate synthase, farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, monoterpene synthase, geranylgeranyl diphosphate reductase, phytoene synthase, and phytoene desaturase. Our results show that liverwort oil bodies, which are organelles bound by a single unit membrane, possess isoprenoid biosynthetic enzymes similar to those found in plastids and the cytosol. We postulate that oil bodies play a dynamic role in cell metabolism in addition to their role as sites of essential oil accumulation and sequestration. The occurrence of such enzymes in different cellular compartments might be due to multiple targeting of gene products to various organelles.

Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae.

The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.

Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches.

In this report, we have shown that the yeast amphiphysin-like protein Rvs167p was localized mainly in small cortical patches throughout the cell in unbudding cells. During budding, the patches were polarized at bud emergence site. During mating, Rvs167p was concentrated at the tip of the shmoo. Rvs167p colocalized with actin patches during yeast vegetative growth and mating. Complete disruption of the actin cytoskeleton using Latrunculin-A did not affect Rvs167p localization in patches throughout the cell. In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected. These observations suggest that Rvs167p may localize the actin cortical complex properly. Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, was required but not sufficient for the correct localization of the protein.

Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast.

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.

Evidence for a branched pathway in the polarized cell division of Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae can choose a bud site in one of two different spatial patterns (axial or bipolar) determined by their mating type. Genes important for bud-site selection have been identified and a linear model describing the hierarchy of these genes was proposed. We have uncovered a new class of genes which is required only for the bipolar pattern. The phenotype of the corresponding mutants coupled with epistasis experiments with some budding mutants already described suggest the existence of specific genes for the bipolar pathway.

Influence of early age at menopause on vertebral bone mass.

Menopause leads to rapid bone loss, mainly as a result of estrogen deficiency superimposed on the age-related linear bone loss. The influence of age at menopause on bone loss is unclear, although early menopause is widely considered a risk factor for osteoporosis. Vertebral bone mineral density (BMD) was measured in 1667 women divided into five groups according to hormonal status and age at menopause. Menopausal status was an independent predictor of BMD in a multiregression analysis, along with current age, years since menopause (YSM), weight, and height. For the same chronologic age (55 years), women with early menopause had a 15% lower BMD and a higher YSM than women whose menopause occurred later ("normal" menopause). After adjusting for the interval since menopause, postmenopausal women with early menopause were found to have lower vertebral BMD than postmenopausal women with normal menopause. Finally, after the age of 60, 66% of the women with early menopause had a BMD that was below the fracture threshold compared to 18% of the women with normal menopause. The results of this cross-sectional study suggest that early menopause is associated with a quantitatively higher bone loss than in women with menopause of later onset and thus constitutes a risk factor for osteoporosis.

Yeast mutants affected in viability upon starvation have a modified phospholipid composition.

Selection of mutations, based on the suppression of rvs161 delta defects, was performed. Ten mutants were obtained, ranged amongst four complementation groups, named SUR1, SUR2, SUR3 and SUR4. All sur mutations also suppress a mutation in another gene, RVS167, indicating that all six genes are involved in the same biological pathway. The sur mutant cells have abnormal morphologies in stationary phase, i.e. dumbbell-like in sur1, sur2 or sur3 strains and multi-budded in sur4 strains. Several phenotypic characteristics of the physiological suppressors as well as the rvs161 delta strain itself led us to analyse the phospholipid composition of the mutants. The assays show an overall decrease of the phospholipid amounts and modifications in the relative contents of some phospholipid classes in sur mutant cells.

Risk factors for hip fracture. MEDOS study: results of the Toulouse Centre.

The development of preventive strategies for hip fractures requires better identification of risk factors. The MEDOS study was designed to study prospectively the incidence of hip fracture in 14 centres from six countries and characterise risk factors. At one centre (Toulouse), data were gathered from questionnaires completed by 386 cases of hip fracture aged over 50 years and 848 age- and sex-matched controls over a 12-month period. Of the 935 variables of the MEDOS questionnaire, 235, grouped into 56 items, were statistically analysed. Odds ratios (and 95% confidence intervals) were estimated for each variable from a multiple stepwise logistic regression model. The population comprised 19.2% men and 80.8% women, with a mean age of 80 +/- 8.8 years; 80% were living in an urban area and 76% with their family. Of the 17 significant variables, moderate excess weight and a high nutritional intake of calcium were associated with a decreased risk of hip fracture. Loss of autonomy, a higher height than normal (> 1SD), and a history of previous fractures significantly increased the risk of fracture. Interestingly, all these variables accounted for only 18% of the risk of hip fracture.

Assessment of the risk of post-menopausal osteoporosis using clinical factors.

OBJECTIVE: We wished to assess the predictive value of the main clinical risk factors for osteoporosis over a low vertebral bone mineral density. DESIGN: A cross-sectional study was made of a cohort of peri and post-menopausal women (mean age, 54 years). PATIENTS: One thousand, five hundred and sixty-five normal white women were selected from among the women referred to our menopause clinic for screening and prevention of osteoporosis. MEASUREMENTS: Each woman had replied to a detailed standardized questionnaire including the main clinical risk factors and had her bone density measured using dual photon absorptiometry. RESULTS: The predictive value for a low vertebral bone mineral density (2 SD below the normal young adult value) was assessed for 15 historical and anthropometric variables. Among these, age, age at menarche, weight, height, menopause and its duration, were independent predictors of a low bone mineral density, in a multiple logistic regression analysis. Odds ratios were calculated for each of these variables, weight, menopause and its duration being the three most influential variables. At best this model makes it possible to correctly classify 73% of women with a low bone mineral density and 66% of those with a normal bone mineral density. If this model is used for screening, it could possibly save 25% of bone densitometry examinations. CONCLUSIONS: Direct bone densitometry remains indispensable to assess osteoporosis risk, since risk factors alone are not sufficient for accurate delineation of either low or normal bone mineral density.

Telematic expert system Diabeto. New tool for diet self-monitoring for diabetic patients.

OBJECTIVE: To evaluate Diabeto, a computer-assisted diet education system. RESEARCH DESIGN AND METHODS: One hundred five patients with insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus (NIDDM) were divided into two randomized groups to participate in the evaluation of Diabeto. With free access through Minitel, the French public videotex network, Diabeto helps diabetic patients self-monitor their diets and balance their meals with personalized counseling. RESULTS: During the first 6-mo study, group A (54 patients) used Diabeto, whereas group B (51 patients) were control subjects. For the second 6-mo study, group B used the system. Evaluation was based on patients' dietetic knowledge, dietary habits, and metabolic balance. CONCLUSIONS: Diabeto led to a significant improvement of dietetic, knowledge in group A (P less than 0.0005) and also to improved dietary habits; decreased caloric intake in patients initially overeating (P less than 0.05), increase of dietary carbohydrate from 39.7 +/- 0.7 to 42.9 +/- 0.9% in patients with an initial intake less than 45% carbohydrate, and decrease of fat intake from 41.9 +/- 0.9 to 37.4 +/- 1.1% in patients with an initial intake of greater than 35% fat (P less than 0.0005). In the second study, in addition to similar improvements to those observed in the first study, HbA1 decreased from 11.0 +/- 0.4 to 9.9 +/- 0.4% (P less than 0.005) and fructosamine from 5.00 +/- 0.17 to 4.57 +/- 0.17% (P less than 0.001). Diabeto appears to be an effective therapeutic tool in the control of metabolic diseases.

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining.

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.

[Risk factors of vertebral osteoporosis. Results of a study of 2279 women referred to a menopause clinic]. / Facteurs de risque de l'ostéoporose vertébrale. Résultats d'une étude de 2,279 femmes adressées à une consultation de ménopause.

The principal clinical risk factors of osteoporosis were correlated with vertebral bone density measured by biphoton absorptiometry in 2,279 women referred to a menopause clinic. Age, age at puberty, the menopause and how long ago it had occurred, smoking and past history of osteoporosis were negatively and independently correlated with bone density. Early onset of the menopause significantly increased the risk of a low bone density as compared with a natural or surgical menopause occurring after the age of 45. In contrast, estrogen replacement therapy and excess weight decreased this risk after the menopause. Osteoarthrosis and 25 (OH) vit. D3 levels were positively correlated with bone density. These variables taken together in a multiple linear regression model were able to explain only 25 per cent of the variations in bone density between individuals, suggesting the predominant role of other factors not taken into account in our study, notably of a genetic and environmental nature. Before the menopause, none of the factors studied enabled prediction of the risk defined by a low bone density. Among women with one or more clinical risk factors correlated with bone density, 29 per cent in fact had low bone density, as compared with 20 per cent in the absence of such factors. In contrast, 54 per cent of women with a low bone density had no risk factor. The results of this study show that the exception of secondary causes of osteoporosis, the majority of so-called "risk" factors which can be assessed by history have only a minimal influence on bone density.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity among the 2 microns plasmids in Saccharomyces cerevisiae: a new sequence for the REP1 gene.

Some species of yeasts contain naturally-occurring circular DNA plasmids. The most studied of these plasmids is the 2 microns circle of Saccharomyces cerevisiae. Three variants of this plasmid, Scp1, Scp2 and Scp3, have been described according to their restriction maps [Cameron et al., Nucleic Acids Res. 4 (1977) 1429-1448; Livingston, Genetics 86 (1977) 73-84]. The entire nucleotide (nt) sequence of the Scp1 variant from strain A364A has been published [Hartley and Donelson, Nature 286 (1980) 860-864]. We report here the nt sequence of the 2 microns plasmid REP1 gene from S. cerevisiae strain SKQ2n. According to the restriction analysis, this plasmid is the Scp3 variant previously described. The only observed differences between the Scp1 and Scp3 variants were the loss of one EcoRI restriction site and an apparent deletion in Scp3. The nt sequence we report differs significantly from the previously published one for Scp1. The differences correspond to 128 (about 8.5%) substituted, deleted or additional nt of 1510 nt compared. These differences affect the coding region (8%) as well as the noncoding regions (9.7%). Regarding the putative encoded proteins, 38 (about 10%) amino acids (aa) are modified or deleted in our sequence and 11 are added. Most of these aa modifications are not randomly distributed but are concentrated in certain regions. These observations are indicative of important intraspecific evolution between the two 2 microns plasmid variants considered, as well as of conservative selection pressure on some domains of the REP1 protein.

Obesity and postmenopausal bone loss: the influence of obesity on vertebral density and bone turnover in postmenopausal women.

This study was undertaken to evaluate the effect of obesity on the postmenopausal bone mass. Bone mineral density, measured by dual photon absorptiometry of the lumbar spine, serum osteocalcin (OC), fasting urinary calcium to creatinine (Ca:Cr), serum estradiol (E2) dehydroepiandrosterone (DHA) and testosterone (T) were measured in 176 women aged 45-71 years. Women were divided into four groups according to their menopausal status and their weight: 49 perimenopausal, 28 obese perimenopausal, 49 obese postmenopausal. Within each population (perimenopausal and postmenopausal), mean age was the same, only weight was significantly different (p less than 0.0001). For the two groups of postmenopausal women mean interval since menopause (YSM) was the same (5.8 +/- 3 and 5.4 +/- 5 yr). Comparison between groups revealed a significant effect of menopausal status and obesity on BMD and bone turnover. As compared to perimenopausal women, BMD was lower, OC and Ca: Cr higher only in nonobese-postmenopausal women. E2, T, DHA did not differ between the two groups of postmenopausal women. The results of this study suggest that even moderate obesity can play a protective role on postmenopausal bone loss.

A New Method for the Screening of Mutations Related to Protoplasmic Incompatibility, Differentiation and Plasma Membrane in the Fungus PODOSPORA ANSERINA.

In Podospora anserina previous investigations showed that mutations in genes involved in the control of protoplasmic incompatibility cause defects at various stages of differentiation during the life cycle and also modify properties of the plasma membrane. To establish these relationships in another way, a new method for screening mutations has been developed as a first step. Eighty-five new mutants were selected for resistance to toxic products (sorbose or thiourea); in a second step these mutants were tested for modifications of protoplasmic incompatibility and cellular differentiation. Seven of the sorbose or thiourea resistant-mutants (i.e., 8%) exhibited new patterns of protoplasmic incompatibility. Genetic analyses were carried out with three mutants. Mutation X25 suppresses protoplasmic incompatibility resulting from all allelic interactions and restores the fertility of the crosses female symbol Vx male symbol V1 and female symbol Z1 x male symbol Z2. Mutation V41 is an allele of the v locus with new properties. Mutation X61 totally suppresses the V/V'1 interaction and weakens all of the other allelic incompatibility systems; X61 strains are defective in protoperithecia differentiation. Electrophoresis of plasma membrane proteins showed that X61 strains lack two polypeptides whose apparent molecular weights are 41,000 and 44,000. This new screening method is especially efficient for obtaining new mutants and identifying additional genes involved in incompatibility. These results provide further support demonstrating the relationships between protoplasmic incompatibility, cellular differentiation and plasma membrane.