The epidemiology and antigenic characterization of influenza viruses isolated in Curitiba, South Brazil

Several studies conducted all over the world have reported that the influenza virus is associated with great morbidity and mortality rates. In this study, we analyzed the incidence of the influenza virus between 2000 and 2003 in Curitiba. We studied 1621 samples obtained from outpatients and hospitalized patients of both sexes and all ages. The study was conducted at the local primary care health units (outpatients) and at the tertiary care unit (hospitalized) of the General Hospital of the Federal University in the state of Paran��, Brazil. Nasopharyngeal aspirates and, eventually, bronchoalveolar lavage were assayed for the presence of viral antigens, either by indirect immunofluorescence or cell culture. Of the samples studied, 135 (8.3%) were positive for influenza virus, and of those, 103 (76.3%) were positive for type A and 32 (23.7%) for type B. Additionally, positive samples were analyzed by reverse transcription followed by polymerase chain reaction and subtypes H1 and H3 were identified from this group. A high incidence of positive samples was observed mainly in the months with lower temperatures. Furthermore, outpatients showed a higher incidence of influenza viruses than hospitalized patients.

Genetic male infertility and mutation of CATSPER ion channels.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression.

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca(2+); (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Use of stains to detect fingermarks

Detection of fi ngermarks at a crime scene or on related items is of prime interest for forensicinvestigators, mainly for identifi cation purposes. Most of the fi ngermarks are invisible to thenaked eye, however. The application of detection techniques is required to establish visual contrastbetween the secretion residue and the underlying substrate. We give here a review of thefi eld related to the concept of using stains to detect fi ngermarks. A distinction has been madebetween the physically driven classical detection techniques, the chemically driven ones, andthose based on nanostructured materials, an emerging fi eld in forensic science.

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart.Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the alpha 1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first-and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity.Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Recepci��n, clasi���caci��n seg��n escala Manchester y atenci��n sanitaria inicial al paciente que acude a un Dispositivo de Cuidados Cr��ticos y Urgencias (DCCU) con motivo de consulta ���Diabetes���: Posibilidad de intervenci��n ���nalista de la enfermera en la consulta de triage (Triage Avanzado) ante la hipoglucemia. Colaboraci��n multidisciplinar y criterios de derivaci��n

The Andalusian Public Health System (SSPA) is considering the last time an attempt urgent process management through triage consultation, both hospital and primary care environment and tables situations in which the nurse responsible for these consultations can carry out a ���nal statement of which only she is directly responsible through their independent intervention and referral (Triage Advanced). Pose, at once and consistently to the idea of teamwork, where they can be the limits to that intervention ���nalist and the circuits to follow. This paper proposes a de���nition line of one of those situations through triage concepts universally tested, and takes full advantage of advanced practice pro���le offered by nurses Device Critical Care (DCCU) of the SSPA and any the emerging legal and regulatory framework in terms of standardized collaborative prescription, us know legitimate receivers. This work stems from the vision of professionals and our contribution to that line of institutional work that must be consensus.

Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target ampli���cation technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Distinct chitinases are expressed during various growth phases of the human pathogen Paracoccidioides brasiliensis

The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

R��sum�� au large public Notre corps est constitu�� de diff��rents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'��nergie. Cette t��che est assum��e en partie par une prot��ine qui se situe dans la membrane de chaque cellule. Nomm�� Na, K��ATPase ou pompe �� sodium, c'est une prot��ine pressente dans toutes les cellules chez les mammif��res est compos��e de deux sous-unit��s, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium �� l'int��rieur de la cellule, elle transforme l'��nergie chimique sous forme de l'ATP en ��nergie motrice, qui permet aux cellules par la suite d'��changer des mat��riaux entre l'espace intracellulaire et extracellulaire ainsi que d'ing��rer des nutriments provenant de son environnement. Le manque de cette prot��ine chez la souris entra��ne la mort de l'embryon. Des d��fauts fonctionnels de cette prot��ine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette prot��ine est ��galement engag��e dans diverses activit��s physiologiques comme la contractilit�� musculaire, l'activit�� nerveuse et la r��gulation du volume sanguin. Vue l'importance de cette prot��ine, sa d��couverte par Jens C. Skou en 1957 a ��t�� honor��e d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les m��canismes de fonctionnement de la Na, K-ATPase. Entre autre, sa r��gulation par une famille de prot��ines appel��es prot��ines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nomm�� FXYD 2 est li�� �� une maladie h��r��ditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les cons��quences de la r��gulation par les prot��ines FXYD sur activit�� de la Na, K-ATPase, mais nous savons tr��s peu sur le mode d'interaction entre les prot��ines FXYD et la Na, K-ATPase. Dans ce travail de th��se, nous avons r��ussi �� localiser des zones d'interaction dans la sous- unit�� a de la Na, K-ATPase et dans FXYD 7. En m��me temps, nous avons d��termin�� un 3��me site de liaison sp��cifique au sodium de la Na, K-ATPase. Une partie de ce site se situe �� l'int��rieur d'un domaine prot��ique qui interagit avec les prot��ines FXYD. De plus, ce site a ��t�� d��montr�� comme responsable d'un m��canisme de transport de la Na, K-ATPase caract��ris�� par un influx ionique. En conclusion, les r��sultats de ce travail de th��se fournissent de nouvelles preuves sur les r��gions d'interaction entre la Na, K-ATPase et les prot��ines FXYD. La d��termination d'un 3��me site sp��cifique au sodium et sa relation avec un influx ionique offrent la possibilit�� 1) d'explorer les m��canismes avec lesquels les prot��ines FXYD r��gulent l'activit�� de la Na, ATPase et 2) de localiser un site �� sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. R��sum�� Les gradients de concentration de Na+ et de K+ �� travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'hom��ostasie de cellules. De plus, des fonctions cellulaires sp��cifiques telles que la reabsorption de Na dans le rein et le c��lon, la contraction musculaire et l'excitabilit�� nerveuse d��pendent de ces gradients. La Na, K��ATPase ou pompe �� sodium est une prot��ine membranaire ubiquitaire. Elle cr��e et maintient ces gradients en utilisant l'��nergie obtenu par l'hydrolyse de l'ad��nosine triphosphate. L'unit�� fonctionnelle minimale de cette prot��ine se compose d'une sous-unit�� catalytique α et d'une sous-unit�� r��gulatrice β. R��cemment, il a ��t�� montr�� que des membres de la famille FXYD, sont des r��gulateurs tissu-sp��cifiques de la Na, K-ATPase qui influencent ses propri��t��s de transport. Cependant, on conna��t peu de chose au sujet de la nature mol��culaire de l'interaction entre les prot��ines FXYD et la Na, K-ATPase. Dans cette ��tude, nous fournissons, pour la premi��re fois, l'��vidence directe que des r��sidus du domaine transmembranaire (TM) 9 de la sous-unit�� α de la Na, K-ATPase sont impliqu��s dans l'interaction fonctionnelle et structurale avec les prot��ines FXYD. De plus nous avons identifi�� des r��gions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une s��rie de r��sidus responsables des r��gulations fonctionnelles. Nous avons aussi montr�� les contributions fonctionnelles du TM 9 de la Na, K-ATPase �� la translocation de Na + en d��terminant un 3��me site sp��cifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unit�� α de la pompe �� sodium. De plus, nous avons constat�� que le 3��me site de Na + est fonctionnellement li�� �� un courant entrant de la pompe sensible �� l'ouaba��ne et activ�� par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les prot��ines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilit��s pour explorer le m��canisme par lequel les prot��ines FXYD r��gulent les propri��t��s fonctionnelles de la Na, K-ATPase. La d��termination du 3��me site au Na + fournit une compr��hension avanc��e du site sp��cifique au Na + de la Na, K-ATPase et du m��canisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Pharmacological administration of the isoflavone daidzein enhances cell proliferation and reduces high fat diet-induced apoptosis and gliosis in the rat hippocampus.

Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ER��) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17��-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ER�� in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.

Analysis of a boundary protein delimiting chromatin domains in yeast

SUMMARY The expression state of a eukaryotic gene depends in part on its location in the chromosome. This position effect results from the organization of eukaryotic genomes into discrete functional domains, defined by local differences in chromatin structure. The expression of genes within each domain appears to be defined and maintained by the concerted action of regulatory elements such as promoters, enhancers, silencers and locus control regions. Individual domains may be bordered by boundary elements that separate regions of permissive and silent chromatin. When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres towards more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one of these transcription factors, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino-acid substitutions that similarly inhibited the boundary and histone-binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains. RESUME L'expression des g��nes eucaryotes d��pend en partie de leur localisation sur les chromosomes. Cet effet de position r��sulte de l'organisation des g��nomes eucaryotes en domaines fonctionnels, d��finis par des changements locaux au niveau de la structure de la chromatine. Dans chacun de ces domaines, l'expression des g��nes est d��finie et maintenue par l'action concert��e de diff��rents ��l��ments r��gulateurs tels que les promoteurs, les amplificateurs, les silenceurs et les locus control r��gions. Ces domaines peuvent ��tre entour��s par des ��l��ments barri��re, s��parant les r��gions de chromatine r��pressive des r��gions permissive pour l'expression des g��nes. Lorsqu'ils se situent �� proximit�� d'��l��ments chromosomiques comme les telom��res, les g��nes peuvent ��tre r��prim��s de mani��re ��pig��n��tique. Chez la levure, cette r��pression est ��tablie par la propagation des prot��ines SIR depuis les t��lom��res vers les r��gions centrom��riques. Certains facteurs de transcription peuvent emp��cher la r��pression d'un g��ne, lorsqu'ils sont plac��s entre ce g��ne et le t��lom��re. Nous avons ��tudi�� un de ces facteurs, CTF-1, qui a la particularit�� de lier directement l'histone H3. La d��l��tion de certaines parties de CTF-1 a permis de d��terminer que la r��gion responsable de l'activit�� barri��re correspond au domaine d'interaction avec H3. Plusieurs mutations points effectu��es dans ce domaine inhibent �� la fois l'activit�� barri��re et la capacit�� de lier H3. Des exp��riences d'immuno-pr��cipitation de la chromatine indiquent que la prot��ine barri��re CTF-1 pr��vient efficacement la propagation des prot��ines SIR et s��pare des domaines contenant des histones hypo-ac��tyl��es de ceux constitu��s d'histones hyper-ac��tyl��es. Ces r��sultats sugg��rent que CTF-1 interagit directement avec l'histone H3 pour emp��cher la propagation de la chromatine r��pressive, d��limitant ainsi des domaines de chromatine permissive et des domaines de chromatine silencieuse.

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

R��sum�� : Les jasmonates (JA), une famille d'hor1none v��g��tale, jouent un r��le central dans la r��ponse �� la blessure, et aux attaques d'insectes et de pathog��nes. Les JA sont principalement d��riv��s d'un acide gras, l'acide linol��nique. L'addition par une lipoxyg��nase d'une mol��cule d'oxyg��ne �� l'acide linol��nique initie la synth��se de JA. Cependant les m��canismes r��gulant l'activation de la biosynth��se de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caract��ris�� chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activit�� lipoxyg��nase est plus ��lev��e que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus ��lev��s et l'activation de la synth��se de JA apr��s blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus r��sistant au pathog��ne Botrytis cinerea et �� la chenille Spodoptera littoralis. Afin de comprendre quel m��canisme chez fou2 g��n��re ce ph��notype, nous avons clon�� le g��ne responsable du ph��notype de fou2. Le mutant fou2 porte une mutation dans le g��ne d'un canal �� deux pores transportant probablement du potassium, du lumen de la vacuole v��g��tale vers le compartiment cytosolique. L'analyse du prot��ome de fou2 a permis d'identifier une expression plus ��lev��e de sept prot��ines r��gul��es par les JA ou le stress. La d��couverte de l'implication d'un canal dans le ph��notype de fou2 renforce l'hypoth��se que les flux de cations pourraient ��tre impliqu��s dans les ��tapes pr��coces de la synth��se des JA. Nous avons ��galement ��tudi�� le prot��ome et la physiologie d'une feuille bless��e, Pour ��valuer les changements d'expression prot��ique en r��ponse �� la blessure et contr��l��s par les JA, nous avons quantifi�� l'expression de 5937 prot��ines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synth��tiser des JA. Parmi ces 5937 prot��ines, nous avons identifi�� 99 prot��ines r��gul��es par la blessure chez le type sauvage. Nous avons observ�� pour 65% des prot��ines dont l'expression prot��ique changeait apr��s blessure une bonne corr��lation entre la quantit�� de transcrits et de prot��ines. Plusieurs enzymes de la voie des chorismates impliqu��es dans la biosynth��se des acides amin��s ph��noliques ��taient induites par les JA apr��s blessure. Une quantification des acides amin��s a montr�� que les niveaux d'acides amin��s ph��noliques augmentaient significativement apr��s blessure. La blessure induisait aussi des changements dans l'expression de prot��ines impliqu��es dans la r��ponse au stress et particuli��rement au stress oxydatif. Nous avons quantifi�� l'��tat r��duit et oxyd�� du glutathion, un tripeptide qui, sous sa forme r��duite, est l'antioxydant majeur des cellules. Nous avons trouv�� une quantit�� significativement plus ��lev��e de glutathion oxyd�� chez le type sauvage bless�� que chez la plante aus bless��e. Ce r��sultat sugg��re que la g��n��ration d'un stress oxydatif et la proportion relative de glutathions r��duits et oxyd��s sont contr��l��s par les JA apr��s blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.

CHD8 associates with human Staf and contributes to efficient U6 RNA polymerase III transcription.

Chromatin remodeling and histone modification are essential for eukaryotic transcription regulation, but little is known about chromatin-modifying activities acting on RNA polymerase III (Pol III)-transcribed genes. The human U6 small nuclear RNA promoter, located 5' of the transcription start site, consists of a core region directing basal transcription and an activating region that recruits the transcription factors Oct-1 and Staf (ZNF143). Oct-1 activates transcription in part by helping recruit core binding factors, but nothing is known about the mechanisms of transcription activation by Staf. We show that Staf activates U6 transcription from a preassembled chromatin template in vitro and associates with several proteins linked to chromatin modification, among them chromodomain-helicase-DNA binding protein 8 (CHD8). CHD8 binds to histone H3 di- and trimethylated on lysine 4. It resides on the human U6 promoter as well as the mRNA IRF3 promoter in vivo and contributes to efficient transcription from both these promoters. Thus, Pol III transcription from type 3 promoters uses some of the same factors used for chromatin remodeling at Pol II promoters.