Role of the JNK-interacting protein 1/islet brain 1 in cell degeneration in Alzheimer disease and diabetes.

Numerous epidemiological studies and some pharmacological clinical trials show the close connection between Alzheimer disease (AD) and type 2 diabetes (T2D) and thereby, shed more light into the existence of possible similar pathogenic mechanisms between these two diseases. Diabetes increases the risk of developing AD and sensitizers of insulin currently used as diabetes drugs can efficiently slow cognitive decline of the neurological disorder. Deposits of amyloid aggregate and hyperphosphorylation of tau, which are hallmarks of AD, have been also found in degenerating pancreatic islets beta-cells of patients with T2D. These events may have a causal role in the pathogenesis of the two diseases. Increased c-Jun NH(2)-terminal kinase (JNK) activity is found in neurofibrillary tangles (NFT) of AD and promotes programmed cell death of beta-cells exposed to a diabetic environment. The JNK-interacting protein 1 (JIP-1), also called islet brain 1 (IB1) because it is mostly expressed in the brain and islets, is a key regulator of the JNK pathway in neuronal and beta-cells. JNK, hyperphosphorylated tau and IB1/JIP-1 all co-localize with amyloids deposits in NFT and islets of AD and patients with T2D. This review discusses the role of the IB1/JIP-1 and the JNK pathway in the molecular pathogenesis of AD and T2D.

Microtubule-associated protein 1B binds glyceraldehyde-3-phosphate dehydrogenase.

Microtubule-associated protein 1B, MAP1B, is a major cytoskeletal protein during brain development and one of the largest brain MAPs associated with microtubules and microfilaments. Here, we identified several proteins that bind to MAP1B via immunoprecipitation with a MAP1B-specific antibody, by one and two-dimensional gel electrophoresis and subsequent mass spectrometry identification of precipitated proteins. In addition to tubulin and actin, a variety of proteins were identified. Among these proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 8, dihydropyrimidinase related proteins 2 and 3, protein-L-isoaspartate O-methyltransferase, beta-spectrin, and clathrin protein MKIAA0034, linking either directly or indirectly to MAP1B. In particular, GAPDH, a key glycolytic enzyme, was bound in large quantity to the heavy chain of MAP1B in adult brain tissue. In vitro binding studies confirmed a direct binding of GAPDH to MAP1B. In PC12 cells, GAPDH was found in cytoplasm and nuclei and partially co-localized with MAP1B. It disappeared from the cytoplasm under oxidative stress or after a disruption of cytoskeletal elements after colcemid or cytochalasin exposure. GAPDH may be essential in the local energy provision of cytoskeletal structures and MAP1B may help to keep this key enzyme close to the cytoskeleton.

Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles.

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo₂ cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo₂ cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Oxidative potential for fine/ultrafine particles in occupational situations

Introduction : The redox properties of fine/ultrafine particles as well as nanoparticles (NP) are suggested to be important to explain their biological activity and could constitute a novel and promising metric for hazard evaluation. The acellular in vitro dithiothreitol (DTT) assay allows measuring this property. Objectives : (1) to evaluate sampling requirements for fine/ultrafine particle allowing measurement of their oxidative potential (2) to apply the methodology to occupational situations where particle from combustion sources are generated. Material and method : Sampling parameters (type of filters and loaded amount) and storage duration affecting the DTT measurements were evaluated. Based on these results, a methodological approach was defined and applied in two occupational situations where diesel and other combustion particles are present (toll station in a tunnel and mechanical yard for bus reparation). Results : Teflon filters loaded with diesel particles were found more suitable for the DTT assay, due to their better chemical inertness compared to quartz filters: after storage durations larger than 150 hours, an increased reactivity toward DTT was observed only with quartz filters. Reactivity was linearly correlated to the loaded mass until about 1000 μg/filter. Different redox reactivities were determined in both working places, with the mechanical yard presenting a higher DTT consumption rate. Discussion and conclusions : These results demonstrate the feasibility of this method to determine the oxidative potential of fine/ultrafine particles in occupational situations. We propose to include this approach for hazard assessment of work places with exposure to manufactured and other NP.

Evidence against a direct role of klotho in insulin resistance.

The klotho gene may be involved in the aging process. Klotho is a coactivator of FGF23, a regulator of phosphate and vitamin D metabolism. It has also been reported to be downregulated in insulin resistance syndromes and paradoxically to directly inhibit IGF-1 and insulin signaling. Our aim was to study klotho's regulation and effects on insulin and IGF-1 signaling to unravel this paradox. We studied klotho tissue distribution and expression by quantitative real-time polymerase chain reaction and Western blotting in obese Zucker rats and high-fat fed Wistar rats, two models of insulin resistance. Klotho was expressed in kidneys but at much lower levels (<1.5%) in liver, muscle, brain, and adipose tissue. There were no significant differences between insulin resistant and control animals. We next produced human recombinant soluble klotho protein (KLEC) and studied its effects on insulin and IGF-1 signaling in cultured cells. In HEK293 cells, FGF23 signaling (judged by FRS2-alpha and ERK1/2 phosphorylation) was activated by conditioned media from KLEC-producing cells (CM-KLEC); however, IGF-1 signaling was unaffected. CM-KLEC did not inhibit IGF-1 and insulin signaling in L6 and Hep G2 cells, as judged by Akt and ERK1/2 phosphorylation. We conclude that decreased klotho expression is not a general feature of rodent models of insulin resistance. Further, the soluble klotho protein does not inhibit IGF-1 and/or insulin signaling in HEK293, L6, and HepG2 cells, arguing against a direct role of klotho in insulin signaling. However, the hypothesis that klotho indirectly regulates insulin sensitivity via FGF23 activation remains to be investigated.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Developmental critical period in gentic redox dysregulation: animal and human studies in schizophrenia

Converging evidence favors an abnormal susceptibility to oxidative stress in schizophrenia. Decreased levels of glutathione (GSH), the major cellular antioxidant and redox regulator, was observed in cerebrospinal-fluid and prefrontal cortex of patients. Importantly, abnormal GSH synthesis of genetic origin was observed: Two case-control studies showed an association with a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease associated genotypes (35% of patients) correlated with decreased GCLC protein, GCL activity and GSH content. Similar GSH system anomalies were observed in early psychosis patients. Such redox dysregulation combined with environmental stressors at specific developmental stages could underlie structural and functional connectivity anomalies. In pharmacological and knock-out (KO) models, GSH deficit induces anomalies analogous to those reported in patients. (a) morphology: spine density and GABA-parvalbumine immunoreactivity (PV-I) were decreased in anterior cingulate cortex. KO mice showed delayed cortical PV-I at PD10. This effect is exacerbated in mice with increased DA from PD5-10. KO mice exhibit cortical impairment in myelin and perineuronal net known to modulate PV connectivity. (b) physiology: In cultured neurons, NMDA response are depressed by D2 activation. In hippocampus, NMDA-dependent synaptic plasticity is impaired and kainate induced g-oscillations are reduced in parallel to PV-I. (c) cognition: low GSH models show increased sensitivity to stress, hyperactivity, abnormal object recognition, olfactory integration and social behavior. In a clinical study, GSH precursor N-acetyl cysteine (NAC) as add on therapy, improves the negative symptoms and decreases the side effects of antipsychotics. In an auditory oddball paradigm, NAC improves the mismatched negativity, an evoked potential related to pre-attention and to NMDA receptors function. In summary, clinical and experimental evidence converge to demonstrate that a genetically induced dysregulation of GSH synthesis combined with environmental insults in early development represent a major risk factor contributing to the development of schizophrenia Conclusion Based on these data, we proposed a model for PSIP1 promoter activity involving a complex interplay between yet undefined regulatory elements to modulate gene expression.

The tumor suppressor p16Ink4a regulates T lymphocyte survival.

In contrast to other cell cycle inhibitors, the tumor suppressor p16Ink4a is not detectable or expressed at very low levels in embryonic and adult mouse tissues, and therefore it has often been considered as a specialized checkpoint protein that does not participate in the control of normal cell cycle progression. However, Ink4a-/- mice possess increased thymus size and cellularity, thus suggesting the involvement of p16(Ink4a) in the control of thymocyte proliferation. In this study, we found increased numbers of CD8 and CD4 T lymphocytes in thymus and spleen from Ink4a-/- mice. Unexpectedly, this was not related to an increase in T-cell division rates, which were similar in lymphoid organs of Ink4a-/- and wild-type mice. In contrast, T-cell apoptosis rates were significantly decreased in thymus and spleen from Ink4a-/- mice. Moreover, whereas p16Ink4a-deficient and wild-type T cells were equally sensitive to Fas or TCR-mediated apoptosis, the former were clearly more resistant to apoptosis induced by oxidative stress or gamma irradiation. Our results indicate that p16Ink4a function is associated with T-cell apoptosis, and subsequently contributes to the control of T-cell population size in lymphoid organs.

Response of the Pacific oyster Crassostrea gigas, Thunberg 1793, to pesticide exposure under experimental conditions.

Pesticide run-off into the ocean represents a potential threat to marine organisms, especially bivalves living in coastal environments. However, little is known about the effects of environmentally relevant concentrations of pesticides at the individual level. In this study, the suppression subtractive hybridisation technique was used to discover the main physiological function affected by a cocktail of three pesticides (lindane, metolachlor and carbofuran) in the Pacific oyster Crassostrea gigas. Two oyster populations exposed to different pollution levels in the wild were investigated. The pesticide concentrations used to induce stress were close to those found in the wild. In a time course experiment, the expression of three genes implicated in iron metabolism and oxidative stress as well as that of two ubiquitous stress proteins was examined. No clear regulation of gene or protein expression was found, potentially due to a low-dose effect. However, we detected a strong site- and organ-specific response to the pesticides. This study thus (1) provides insight into bivalve responses to pesticide pollution at the level of the transcriptome, which is the first level of response for organisms facing pollution, and (2) raises interesting questions concerning the importance of the sites and organs studied in the toxicogenomic field.

Involvement of environmental mercury and lead in the etiology of neurodegenerative diseases.

The incidence of neurodegenerative disease like Parkinson's disease and Alzheimer's disease (AD) increases dramatically with age; only a small percentage is directly related to familial forms. The etiology of the most abundant, sporadic forms is complex and multifactorial, involving both genetic and environmental factors. Several environmental pollutants have been associated with neurodegenerative disorders. The present article focuses on results obtained in experimental neurotoxicology studies that indicate a potential pathogenic role of lead and mercury in the development of neurodegenerative diseases. Both heavy metals have been shown to interfere with a multitude of intracellular targets, thereby contributing to several pathogenic processes typical of neurodegenerative disorders, including mitochondrial dysfunction, oxidative stress, deregulation of protein turnover, and brain inflammation. Exposure to heavy metals early in development can precondition the brain for developing a neurodegenerative disease later in life. Alternatively, heavy metals can exert their adverse effects through acute neurotoxicity or through slow accumulation during prolonged periods of life. The pro-oxidant effects of heavy metals can exacerbate the age-related increase in oxidative stress that is related to the decline of the antioxidant defense systems. Brain inflammatory reactions also generate oxidative stress. Chronic inflammation can contribute to the formation of the senile plaques that are typical for AD. In accord with this view, nonsteroidal anti-inflammatory drugs and antioxidants suppress early pathogenic processes leading to Alzheimer's disease, thus decreasing the risk of developing the disease. The effects of lead and mercury were also tested in aggregating brain-cell cultures of fetal rat telencephalon, a three-dimensional brain-cell culture system. The continuous application for 10 to 50 days of non-cytotoxic concentrations of heavy metals resulted in their accumulation in brain cells and the occurrence of delayed toxic effects. When applied at non-toxic concentrations, methylmercury, the most common environmental form of mercury, becomes neurotoxic under pro-oxidant conditions. Furthermore, lead and mercury induce glial cell reactivity, a hallmark of brain inflammation. Both mercury and lead increase the expression of the amyloid precursor protein; mercury also stimulates the formation of insoluble beta-amyloid, which plays a crucial role in the pathogenesis of AD and causes oxidative stress and neurotoxicity in vitro. Taken together, a considerable body of evidence suggests that the heavy metals lead and mercury contribute to the etiology of neurodegenerative diseases and emphasizes the importance of taking preventive measures in this regard.

Identification of small RNAs in Pseudomonas species

Summary: Bacterial small RNAs (sRNAs) are transcripts most of which have regulatory functions. Sequence and secondary structure elements enable numerous sRNAs to interact with mRNAs or with regulatory proteins resulting in diverse regulatory effects on virulence, iron storage, organization of cell envelope proteins or stress response. sRNAs having high affinity for RsmA-like RNA-binding proteins are important for posttranscriptional regulation in various Gram-negative bacteria. In Pseudomonas spp., the GacS/GacA two component system positively controls the production of such sRNAs. They titrate RsmA-like proteins and thus overcome translational repression due to these proteins. As a consequence, secondary metabolites can be produced that are implicated in the biocontrol capacity of P. fluorescens or in the virulence of P. aeruginosa. A genome-wide search carried out in P. aeruginosa PAO1 and in closely related Pseudomonas spp. resulted in the identification of 15 genes coding for sRNAs. Eight of these are novel, the remaining seven have previously been observed. Among them, the 1698 sRNA gene was expressed under GacA control, whereas the transcription of 1887 sRNA gene was transcribed under the control of the anaerobic regulator Anr in an oxygen-limited environment. Overexpression of 1698 sRNA in P. fluorescens strain CHAO did not affect the expression of the GacA-regulated hcnA gene (first gene of the operon coding for HCN synthase), indicating that 1698 sRNA is probably not part of the secondary metabolite regulation pathway. The expression of 1698 sRNA was positively regulated by RpoS in both P. aeruginosa PAO 1 and P. ,fluorescens CHAO and appeared to be modulated temporarily by oxidative stress conditions. However, the effect of 1698 sRNA on oxidative stress survival has not yet been established. Hfq protein interacted with 1698 sRNA in vitro and improved 1698 sRNA expression in vivo in P. aeruginosa. In P. fluorescens, GacA and Hfq were both required for expression of rpoS and GacA showed a positively control on the hfq expression; therefore, at least in this organism, GacA control of 1698 sRNA expression may act indirectly via Hfq and RpoS. Different methods were employed to find abase-pairing target for 1698 sRNA. In a proteomic analysis carried out in P. aeruginosa, positive regulation by 1698 sRNA was observed for Soda, the iron-associated superoxide dismutase, an enzyme involved in oxidative stress resistance. A sequence complementary with 1698 sRNA was predicted to be located in the 5' leader of soda mRNA. However, base-pairing between soda mRNA and 1698 sRNA remains to be proven. In conclusion, this work has revealed eight novel sRNAs and novel functions of two sRNAs in Pseudomonas spp. Résumé Les petits ARNs non-codants (sRNAs) produits par les bactéries sont des transcrits ayant pour la plupart des activités régulatrices importantes. Leurs séquences nucléotidiques ainsi que leurs structures secondaires permettent aux sRNAs d'interagir soit avec des RNA messagers (mRNAs), de sorte à modifier l'expression des protéines pour lesquelles ils codent, soit avec des protéines régulatrices liant des rnRNAs, ce qui a pour effet de modifier l'expression de ces mRNAs. Des sRNAs sont impliqués dans diverses voies de régulation, telles que celles qui régissent la virulence, le stockage du fer, l'organisation des protéines de l'enveloppe bactérienne ou la réponse au stress. Chez les Pseudomonas spp., le système à deux composantes GacS/GacA contrôle la production de métabolites secondaires. Ceux-ci sont engagés dans l'établissement du biocontrôle, chez P. fluorescens, ou. de la virulence, chez P. aeruginosa. La régulation génique dirigée par le système GacS/GacA fait intervenir les sRNAs du type RsmZ, capables de contrecarrer l'action au niveau traductionnel exercée par les protéines régulatrices du type RsmA. Une recherche au niveau du génome a été menée chez P. aeruginosa PAO1 de même que chez des espèces qui lui sont étroitement apparentées, débouchant sur la mise en évidence de 15 gènes codant pour des sRNAs. Parmi ceux-ci, huit ont été découverts pour la première fois et sept confirment des travaux publiés. L'expression du gène du sRNAs 1698 s'avère être régulée par GacA, vraisemblablement de manière indirecte. La transcription du gène du sRNA 1887 montre une dépendance envers Anr, régulateur de l'anaérobiose, et envers une carence en oxygène. La surexpression du sRNA 1698 chez P. fluorescens CHAO n'affecte pas l'expression de hcnA, un gène du régulon GacA, laissant supposer que le sRNA n'intervient pas dans la régulation des métabolites secondaires. Chez P. aeruginosa PAOI et chez P. fluorescens CHAO, RpoS, le facteur sigma du stress, est nécessaire à l'expression du sRNA 1698, et la concentration de ce dernier est modulée par des conditions de stress oxydatif. Toutefois, un effet du sRNA 1698 quant à la survie suite au stress oxydatif n'a pas été établi. Par ailleurs, l'interaction entre le sRNA 1698 et Hfq, la protéine chaperone de RNAs, in vitro ainsi qu'un rôle positif de Hfq pour l'expression du sRNA 1698 in vivo ont été démontrés chez P. aeruginosa. L'induction de l'expression par GacA de rpoS et de hfq a été confirmée chez P. fluorescens CHAO, suggérant que la régulation par GacA du sRNA 1698 pourrait se faire par l'intermédiaire de RpoS et Hfq. Diverses méthodes ont été employées pour identifier un transcrit qui puisse être apparié par le sRNA 1698. Une analyse de protéome chez P. aeruginosa montre que l'expression de Soda, la superoxyde dismutase associée au fer, est positivement régulée par le sRNA 1698. Soda est une enzyme impliquée dans la résistance au stress oxydatif. Une séquence de complémentarité avec le sRNA 1698 a bien été prédite sur le leader 5' du mRNA de soda. Cependant, l'appariement entre le sRNA et son transcrit cible est encore à prouver. En conclusion, ce travail a dévoilé huit nouveaux sRNAs et de nouvelles fonctions pour deux sRNAs chez les Pseudomonas.

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.