Berry fruits modulate kidney dysfunction and urine metabolome in Dahl salt-sensitive rats.

Berries are rich sources of (poly)phenols which have been associated with the prevention of cardiovascular diseases in animal models and in human clinical trials. Recently, a berry enriched diet was reported to decrease blood pressure and attenuate kidney disease progression on Dahl salt-sensitive rats. However, the relationship between kidney function, metabolism and (poly)phenols was not evaluated. We hypothesize that berries promote metabolic alterations concomitantly with an attenuation of the progression of renal disease. For that, kidney and urinary metabolomic changes induced by the berry enriched diet in hypertensive rats (Dahl salt-sensitive) were analyzed using liquid chromatography (UPLC-MS/MS) and 1H NMR techniques. Moreover, physiological and metabolic parameters, and kidney histopathological data were also collected. The severity of the kidney lesions promoted in Dahl rats by a high salt diet was significantly reduced by berries, namely a decrease in sclerotic glomeruli. In addition, was observed a high urinary excretion of metabolites that are indicators of alterations in glycolysis/gluconeogenesis, citrate cycle, and pyruvate metabolism in the salt induced-hypertensive rats, a metabolic profile counteracted by berries consumption. We also provide novel insights that relates (poly)phenols consumption with alterations in cysteine redox pools. Cysteine contribute to the redox signaling that is normally disrupted during kidney disease onset and progression. Our findings provide a vision about the metabolic responses of hypertensive rats to a (poly)phenol enriched diet, which may contribute to the understanding of the beneficial effects of (poly)phenols in salt-induced hypertension.

Mitochondrion: A Common Organelle for Distinct Cell Deaths?

Mitochondria are deeply involved in cell fate decisions via their multiple roles in metabolism, cell growth, and cell death. In healthy cells, these functions are highly regulated to provide sufficient energy for cell function, maintain cell homeostasis, and avoid undesirable cell death. This is achieved by an orchestrated cooperation of cellular and molecular mechanisms such as mitochondrial mass control (mitophagy vs biogenesis), oxidative phosphorylation, redox and calcium homeostasis, and the balance between pro- and antiapoptotic proteins. In the 1990s, mitochondria have been demonstrated to directly control some forms of regulated cell death as well indirectly through energetic metabolism modulation. However, a large body of literature revealed that distinct cell death modalities can coexist in vivo as well as that mitochondria can be dispensable for certain forms of cell death. Likewise, unexpected interconnections between cell death pathways can lead to an amplification of lethality, as well as a defeat of cell death resistance mechanisms. This revealed a complexity of the control of cell fate and a crucial need to reevaluate the role of mitochondria. Here, we will review the various cell death pathways such as apoptosis and mitochondrial permeability transition-driven necrosis and discuss how mitochondrial proteins and mitophagy regulate them. Finally, the role of mitochondrial proteins in the triggering of cell death and mitophagy in pathological models, such as cardiac and brain pathologies, will be highlighted. This may help to define efficient cytoprotective therapeutic strategies based on the targeting of mitochondria.

Non-invasive biological quantification of acute gastrointestinal graft-versus-host disease in children by plasma citrulline.

Clinical grading of GI involvement during acute GVHD remains a challenging issue, especially in children. Plasma citrulline, a non-protein amino acid selectively produced and released by enterocytes, is a suitable surrogate endpoint for small intestinal epithelial cell mass, irrespective of the underlying cause of cell loss. Children referred for allogeneic bone marrow transplantation who were free from chronic malabsorption or constitutional disease involving the GI tract were consecutively included in this prospective study. Plasma citrulline and albumin concentration was measured every week between day 7 and day 28 of BMT until resolution of the aGVHD or occurrence of chronic GVHD. In total, 31 children were included between 2008 and 2011. After a CR, citrulline levels fell to a minimum level on day 7 and then increased to reach the initial value on day 28. After day 28, plasma citrulline but not albumin was strongly linked to the occurrence of GI GVHD, the threshold being set at 10 µmol/L. The correlation with clinical grade of GI-aGVHD now needs to be assessed in larger populations. In pediatric patients, citrulline is valuable as a suitable non-invasive marker of GI involvement in acute GVHD.

[Sodium restriction prevents cardiovascular remodeling associated with insulin-resistance in the rat]. / La restriction sodée prévient le remodelage cardiovasculaire dans l'insulinorésistance chez le rat.

AIM OF THE STUDY: In the present work, the objective was to evaluate the influence of a dietary sodium restriction on cardiovascular morphology changes associated with insulin-resistance. ANIMALS AND PROTOCOL: At 8 weeks of age, rats were fed for 12 weeks a 60%-fructose diet containing a regular sodium content (0.64%) or totally lacking in sodium chloride (<0.01%). A group of rats fed a wheat starch-based diet with regular sodium content served as control group. RESULTS: Elevated HOMA index and plasma insulin confirm the presence of insulin-resistance in fructose-fed rats. Concomitantly, an increase in cardiac mass and in cardiac collagen (Sirius red staining) was detected without obvious change in arterial pressure or cardiac aldosterone synthase mRNA expression. In addition, cross-sectional area of the carotid artery was higher in fructose-fed rats. Production of superoxide anion, equated with dihydroethidium (DHE) staining, was enhanced in cardiac tissue of rats with insulin-resistance. Withdrawal of sodium from the fructose diet prevented all the cardiovascular effects of fructose consumption, including DHE staining. CONCLUSION: These results are in favor of the participation of oxidative stress normalization in the beneficial influence of dietary sodium deprivation on cardiovascular remodeling in this model of insulin-resistance in rats.

[Oxydative stress and beneficial effect of sodium restriction on kidney damage associated with insulin resistance in rats]. / Stress oxydant et effets bénéfiques rénaux de la restriction sodée dans l'insulinorésistance chez le rat.

The aim of this work was to evaluate the influence of dietary sodium restriction on metabolic and renal changes associated with insulin resistance. At 8 weeks of age, rats received either a diet containing 60% fructose with or without sodium or a standard diet for 12 weeks. The insulin resistance and albuminuria induced by the high fructose diet were associated with a fibrosis and increase in oxidative stress in the kidney. The low salt diet prevented insulin resistance, renal fibrosis and albuminuria induced by the fructose diet. These beneficial effects on the kidney were associated with a decrease in kidney NADPH oxidase activity. Oxidative status is probably one of the major targets of the favourable effect of salt restriction on renal changes associated with insulin resistance, without excluding the involvement of other mechanisms.

[Deadly nightshade (Atropa belladonna) intoxication in a 2-year-old child]. / Un cas d'intoxication aiguë par la belladone (Atropa belladona) chez une enfant de 2ans.

Plant intoxications account for 5% of all intoxication cases according to French anti-poison centers. We report an uncommon case of intoxication with deadly nightshade (Atropa belladonna) in a 2-year-old child. The child presented at the ER with an atropinic syndrome, both central and peripheral, after ingestion of wild berries a few hours before. The fruit and leaves brought in by the mother allowed the anti-poison center to identify belladonna, in agreement with clinical findings. The child was kept in the intensive care unit for 48 h and progression was favorable with symptomatic treatment.

Parental comprehension and satisfaction in informed consent in paediatric clinical trials: a prospective study on childhood leukaemia.

OBJECTIVE: To evaluate the extent to which parents are satisfied with and understand the information they are given when their consent is sought for their child to participate in a phase III randomised clinical trial and the reasons for their decision. PATIENTS AND METHOD: The authors carried out a prospective study. The authors included all parents whose consent was sought for their child to participate in the FRALLE 2000A protocol (acute lymphoblastic leukaemia) at two centres. The parents were questioned twice by a qualified psychologist using a semidirected interview, 1 and 6 months after consent was sought. RESULTS: 43 first interviews were carried out. All the parents declared they were satisfied with the explanations provided by the physician. 35 (81%) parents felt that the information provided with the request for consent was appropriate. Eight (19%) parents did not realise that their child had been included in a research protocol. 16 (39%) parents did not understand the concept of randomisation. Half the parents could explain neither the aim of the clinical trial nor the potential benefit of inclusion to their child. Only one third of the parents were aware that they had an alternative. The principal factor underlying their decision, as stated by 29 parents (67%), was confidence in the medical team. CONCLUSIONS: The parents signed consent forms without having fully understood all the elements specific to the experimental protocol. Rather, the parents based their decision on their confidence in the medical team, even when their child's life was at risk.

Abnormal vascular reactivity at rest and exercise in obese boys.

BACKGROUND: Obese children exhibit vascular disorders at rest depending on their pubertal status, degree of obesity, and level of insulin resistance. However, data regarding their vascular function during exercise remain scarce. The aims of the present study were to evaluate vascular morphology and function at rest, and lower limb blood flow during exercise, in prepubertal boys with mild-to-moderate obesity and in lean controls. MATERIALS AND METHODS: Twelve moderately obese prepubertal boys [Body Mass Index (BMI: 23.9+/-2.6 kg m(-2))] and thirteen controls (BMI:17.4+/-1.8 kg m(-2)), matched for age (mean age: 11.6+/-0.6 years) were recruited. We measured carotid intima-media thickness (IMT) and wall compliance and incremental elastic modulus, resting brachial flow-mediated dilation (FMD) and nitrate-dependent dilation (NDD), lower limb blood flow during local knee-extensor incremental and maximal exercise, body fat content (DEXA), blood pressure, blood lipids, insulin and glucose. RESULTS: Compared to lean controls, obese boys had greater IMT (0.47+/-0.06 vs. 0.42+/-0.03 mm, P<0.05) but lower FMD (4.6+/-2.8 vs. 8.8+/-3.2%, P<0.01) in spite of similar maximal shear rate, without NDD differences. Lower limb blood flow (mL min(-1).100 g(-1)) increased significantly from rest to maximal exercise in both groups, although obese children reached lower values than lean counterparts whatever the exercise intensity. CONCLUSIONS: Mild-to-moderate obesity in prepubertal boys without insulin resistance is associated with impaired endothelial function and blunted muscle perfusion response to local dynamic exercise without alteration of vascular smooth muscle reactivity.

The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases.

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].

Cra-dependent transcriptional activation of the icd gene of Escherichia coli.

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli.

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit. The alpha[L262A], alpha[R265A] and alpha[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.

Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family.

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.

DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

[Chlamydia trachomatis infection: risk factors]. / L'infection à Chlamydia trachomatis: facteurs de risques.

UNLABELLED: Genital Chlamydia Trachomatis (CT) infections are the most common cause of tubal infertility. Its prevalence is variable between the authors, but seems to be more frequent in the young population. OBJECTIVE: Evaluate the CT genital infection rate in a population consulting either in a sexually transmitted disease center (STD) or in a family planning unit (FPU). --Judge the interest of a routine screening, and by which technic. --Estimate the information level of population about STD and their prevention. METHODS: 270 men and 331 women were detected by uretral or endocervix sample with an immunofluorescence method (Syva Microtrack). CT serology were also realised. RESULTS: 18.5% of man samples, 20% of woman samples in the STD center and 17.9% of woman samples in the FPU were positive for CT. There is a strong correlation between CT positivity and cervical discharge or cervicitis (RR = 4.64, p < 0.01). Serology was positive in 45.5%, 32.5% and 21.2% respectively for STD center's men, STD center's women and PFU's women. The correlation between samples and serology was 62%. Positive samples were more frequent in the 15-30 years old population and positive serology in the over 30 years old population (RR = 3.57, p < 0.01). There is a correlation between CT positive samples and nulliparity, multiple sexual partner and history of genital infection. CONCLUSION: The high prevalence of CT genital infection observed in our centers justify a routine screening especially in the young population.