Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and ofgrx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.

The monocarboxylate transporter MCT4 : mechanism of regulation in astrocytes and its putative role in cerebral ischemia

Despite its small fraction of the total body weight (2%), the brain contributes for 20% and 25% respectively of the total oxygen and glucose consumption of the whole body. Indeed, glucose has been considered the energy substrate par excellence for the brain. However, evidence accumulated over the last half century revealed an important role for the monocarboxylate lactate in fulfilling the energy needs of neurons. This is particularly true during physiological neuronal activation and in pathological conditions. Lactate transport into and out of the cell is mediated by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, only three of them have been well characterized: MCT2 is the predominant neuronal isoform, while the other non��neuronal cell types of the brain express the ubiquitous isoform MCT1. Quite recently, the MCT4 isoform has been described in astrocytes. Due to its high transport capacity compared to the other two isoforms, MCT4 is particularly adapted for glycolytic cells. Because of its recent discovery in the brain, nothing was known about its regulation in the central nervous system. Here we show that MCT4 is regulated by oxygen levels in primary cultures of astrocytes in a time- and concentration-dependent manner via the hypoxia inducible factor-la (HIF-la). Moreover, we showed that MCT4 expression is essential for astrocyte survival under low oxygen conditions. In parallel, we investigated the possible implication of the pyruvate kinase isoform Pkm2, a strong enhancer of glycolysis, in its regulation. Then we showed that MCT4 expression, as well as the expression of the other two MCT isoforms, is altered in a murine model of stroke. Surprisingly, neurons started to express MCT4, as well as MCT1, under such conditions. Altogether, these data suggest that MCT4, due to its high transport capacity for lactate, may be the isoform that enables cells to operate a major metabolic adaptation in response to pathological situations that alter metabolic homeostasis of the brain. -- Le cerveau repr��sente 2% du poids corporel total, mais il contribue pour 20% de la consommation totale d'oxyg��ne et 25% de celle de glucose au repos. Le glucose est consid��r�� comme le substrat ��nerg��tique par excellence pour le cerveau. N��anmoins, depuis un demi- si��cle maintenant, de plus en plus de travaux ont d��montr�� que le lactate joue un r��le majeur dans le m��tabolisme c��r��bral et est capable du subvenir aux besoins ��nerg��tiques des neurones. Le lactate est tout particuli��rement n��cessaire pendant l'activation neuronale ainsi qu'en situation pathologique. Le transport du lactate �� travers la barri��re h��matoenc��phalique ainsi qu'�� travers les membranes cellulaires est assur�� par la famille des transporteurs aux monocarboxylates (MCTs). Dans le syst��me nerveux central, uniquement trois d'entre eux ont ��t�� d��crits: MCT2 est consid��r�� comme le transporteur neuronal, alors que les autres types cellulaires qui constituent le cerveau expriment l'isoforme ubiquitaire MCT1. R��cemment, l'isoforme MCT4 a ��t�� rapport��e sur les astrocytes. D�� �� sa grande capacit�� de transport pour le lactate, MCT4 est tout particuli��rement adapt�� pour soutenir le m��tabolisme des cellules hautement glycolytiques, comme les astrocytes. En raison de sa toute r��cente d��couverte, les aspects comprenant sa r��gulation et son r��le dans le cerveau sont pour l'instant m��connus. Les r��sultats expos��s dans ce travail d��montrent dans un premier temps que l'expression de MCT4 est r��gul��e par les niveaux d'oxyg��ne dans les cultures d'astrocytes corticaux par le biais du facteur de transcription HIF-la. De plus, nous avons d��montr�� que l'expression de MCT4 est essentielle �� la survie des astrocytes quand le niveau d'oxyg��nation baisse. En parall��le, des r��sultats pr��liminaires sugg��rent que l'isoforme 2 de la pyruvate kinase, un puissant r��gulateur de la glycolyse, pourrait jouer un r��le dans la r��gulation de MCT4. Dans la deuxi��me partie du travail nous avons d��montr�� que l'expression de MCT4, ainsi que celle de MCT1 et MCT2, est alt��r��e dans un mod��le murin d'isch��mie c��r��brale. De fa��on surprenante, les neurones expriment MCT4 dans cette condition, alors que ce n'est pas le cas en condition physiologique. En tenant compte de ces r��sultats, nous sugg��rons que MCT4, d�� �� sa particuli��rement grande capacit�� de transport pour le lactate, repr��sente le MCT qui permet aux cellules du syst��me nerveux central, notamment les astrocytes et les neurones, de s'adapter �� de tr��s fortes perturbations de l'hom��ostasie m��tabolique du cerveau qui surviennent en condition pathologique.

The Saccharomyces cerevisiae Hot1p regulated gene YHR087W (HGI1) has a role in translation upon high glucose concentration stress.

Background While growing in natural environments yeasts can be affected by osmotic stress provoked by high glucose concentrations. The response to this adverse condition requires the HOG pathway and involves transcriptional and posttranscriptional mechanisms initiated by the phosphorylation of this protein, its translocation to the nucleus and activation of transcription factors. One of the genes induced to respond to this injury is YHR087W. It encodes for a protein structurally similar to the N-terminal region of human SBDS whose expression is also induced under other forms of stress and whose deletion determines growth defects at high glucose concentrations. Results In this work we show that YHR087W expression is regulated by several transcription factors depending on the particular stress condition, and Hot1p is particularly relevant for the induction at high glucose concentrations. In this situation, Hot1p, together to Sko1p, binds to YHR087W promoter in a Hog1p-dependent manner. Several evidences obtained indicate Yhr087wp"s role in translation. Firstly, and according to TAP purification experiments, it interacts with proteins involved in translation initiation. Besides, its deletion mutant shows growth defects in the presence of translation inhibitors and displays a slightly slower translation recovery after applying high glucose stress than the wild type strain. Analyses of the association of mRNAs to polysome fractions reveals a lower translation in the mutant strain of the mRNAs corresponding to genes GPD1, HSP78 and HSP104. Conclusions The data demonstrates that expression of Yhr087wp under high glucose concentration is controlled by Hot1p and Sko1p transcription factors, which bind to its promoter. Yhr087wp has a role in translation, maybe in the control of the synthesis of several stress response proteins, which could explain the lower levels of some of these proteins found in previous proteomic analyses and the growth defects of the deletion strain. Keywords: Saccharomyces cerevisiae; High glucose osmotic stress; Gene YHR087W; Gene expression; Translation; Hot1p; Hog1p; Polysomes

Customer expectation study in product development process and its role in strategy formulation

T��m�� ty�� on tehty kansainv��liselle mets��teollisuusyritykselle. Ty��n l��ht��kohtana oli tarve tutkia yrityksen mahdollisuuksia laajentaa toimintaansa uudelle markkinasegmentille, el��inruokapakkausmarkkinoille. Johtuen maailmanlaajuisesta el��inruoankulutuksen kasvusta, el��inruokapakkaukset ovat yritykselle mielenkiintoinen uusi markkina-alue. Onnistunut markkinoilletulo vaatii asiakastarpeiden ja -odotusten huomioimista tuotekehitys- ja markkinastrategian muodostamisessa. T��m��n ty��n tavoitteena oli selvitt���� asiakkaiden odotuksia el��inruokapakkauksille, sek�� mallintaa alalla vallitsevia ostoprosesseja. Asiakastarvetutkimus suunniteltiin perustuen kirjallisuuteen ja alustaviin markkina- ja asiakasanalyyseihin. Alustavat analyysit ovat tarpeellisia l��ht��tilanteen ja trendien selvitt��miseksi. Itse tutkimus toteutettiin semi-konstruktoituna teemahaastatteluna. Haastateltavat edustivat USA:n ja Euroopan johtavia el��inruokavalmistajia ja pakkausten jatkojalostajia. Ty��n tuloksena saatiin yksityiskohtaista tietoa asiakkaiden odotuksista el��inruokapakkauksille, paperin ominaisuuksille ja paperitoimittajille. Lis��ksi mallinnettiin ostoprosesseja paperitoimittajien, pakkausten jalostajien ja el��inruokavalmistajien v��lill��. Tulosten perusteella on analysoitu yrityksen mahdollisuuksia menesty�� uudella markkinasegmentill��. Lis��ksi ty��ss�� annettiin ehdotuksia tuotekehitys- ja markkinastrategian muodostamiseen.

Qualitative social differentiation in tertiary education in Spain

[cat] En aquest treball, s'estudia si la hip��tesi de la desigualtat efectivament mantinguda ���effectively maintained inequality��� es cumpleix a Espanya. Es postula que en les transicions terci��ries a Espanya, les influ��ncies dels pares no es manifesten tant a trav��s de la probabilitat de fer la transici��, sin�� m��s aviat a trav��s de les difer��ncies qualitatives associades a aquesta transici�� en termes de les qualitats dels programes educatius. Analitzem dues caracter��stiques qualitatives dels estudis universitaris: la durada del programa i el seu prestigi acad��mic. Identifiquem per a quins individus la influ��ncia parental ��s m��s important en cada cas.

Qualitative social differentiation in tertiary education in Spain

[cat] En aquest treball, s'estudia si la hip��tesi de la desigualtat efectivament mantinguda ���effectively maintained inequality��� es cumpleix a Espanya. Es postula que en les transicions terci��ries a Espanya, les influ��ncies dels pares no es manifesten tant a trav��s de la probabilitat de fer la transici��, sin�� m��s aviat a trav��s de les difer��ncies qualitatives associades a aquesta transici�� en termes de les qualitats dels programes educatius. Analitzem dues caracter��stiques qualitatives dels estudis universitaris: la durada del programa i el seu prestigi acad��mic. Identifiquem per a quins individus la influ��ncia parental ��s m��s important en cada cas.

Accuracy and Precision of Head Motion Information in Multi-Channel Free Induction Decay Navigators for Magnetic Resonance Imaging.

Free induction decay (FID) navigators were found to qualitatively detect rigid-body head movements, yet it is unknown to what extent they can provide quantitative motion estimates. Here, we acquired FID navigators at different sampling rates and simultaneously measured head movements using a highly accurate optical motion tracking system. This strategy allowed us to estimate the accuracy and precision of FID navigators for quantification of rigid-body head movements. Five subjects were scanned with a 32-channel head coil array on a clinical 3T MR scanner during several resting and guided head movement periods. For each subject we trained a linear regression model based on FID navigator and optical motion tracking signals. FID-based motion model accuracy and precision was evaluated using cross-validation. FID-based prediction of rigid-body head motion was found to be with a mean translational and rotational error of 0.14��0.21 mm and 0.08��0.13(��) , respectively. Robust model training with sub-millimeter and sub-degree accuracy could be achieved using 100 data points with motion magnitudes of ��2 mm and ��1(��) for translation and rotation. The obtained linear models appeared to be subject-specific as inter-subject application of a "universal" FID-based motion model resulted in poor prediction accuracy. The results show that substantial rigid-body motion information is encoded in FID navigator signal time courses. Although, the applied method currently requires the simultaneous acquisition of FID signals and optical tracking data, the findings suggest that multi-channel FID navigators have a potential to complement existing tracking technologies for accurate rigid-body motion detection and correction in MRI.

The central histaminergic neurons in vitro, and their neuroprotective role in excitotoxic cell death in the postnatal rat hippocampus.

Histamine acts as a neurotransmitter in the central nervous system. Brain histamine in synthesized in neurons located to the posterior hypothalamus, from where these neurons send their projections to different parts of the brain. Released histamine participates in the regulation of several physiological functions such as arousal, attention and body homeostasis. Disturbances in the histaminergic system have been detected in diseases such as epilepsy, sleep disorders, anxiety, depression, Alzheimer���s disease, and schizophrenia. The purpose of this thesis was to develop optimal culture conditions for the histaminergic neurons, to study their detailed morphology, and to find out their significance in the kainic acid (KA)-induced neuronal death in the immature rat hippocampus. The morphology of the histaminergic neurons <i>in vitro</i> was comparable with the earlier findings. Histamine-containing vesicles were found in the axon but also in the cell body and dendrites suggesting a possibility for the somatodendritic release. Moreover, histamine was shown to be colocalized with the vesicular monoamine transporter 2 (VMAT2) suggesting that VMAT2 transports histamine to the subcellular storage vesicles. Furthermore, histamine was localized with ��-aminobutyric acid (GABA) in distinct storage vesicles and with neuropeptide galanin partly in the same storage vesicles suggesting different corelease mechanisms for GABA and galanin with histamine. In the organotypic hippocampal slice cultures, KA-induced neuronal death was first detected 12 h after the treatment being restricted mainly to the CA3 subregion. Moreover, cell death was irreversible, since the 48 h recovery period did not save the cells, but instead increased the damage. Finally, neuronal death was suggested to be necrotic, since intracellular apoptotic pathways were not activated, and the morphological changes detected with the electron microscopy were characteristic for necrosis. In the coculture system of the hippocampal and posterior hypothalamic slices, histaminergic neurons significantly decreased epileptiform burst activity and neuronal death in the hippocampal slices, this effect being mediated by histamine 1 (H1) and 3 (H3) receptors. In conclusion, the histaminergic neurons were maintained succesfully in the <i>in vitro</i> conditions exhibiting comparable morphological characteristics as detected earlier <i>in vivo</i>. Moreover, they developed functional innervations within the hippocampal slices in the coculture system. Finally, the KA-induced regionspecific, irreversible and necrotic hippocampal pyramidal cell damage was significantly decreased by the histaminergic neurons through H1 and H3 receptors.

High mobility group box 1 protein (HMGB1) : a pathogenic role in preeclampsia

Introduction : la Physiopathologie maternelle de la pr����clampsie s'associe typiquement �� un ��tat inflammatoire syst��mique mod��r��. La prot��ine "high mobility group box 1" (HMGB-1) est une prot��ine nucl��aire ubiquitaire. En cas de stress cellulaire, elle est rel��ch��e dans le milieu extrace llua li re et peut ainsi exercer son activit�� pro-inflammatoire. En cas de pr����clampsie, le liquide amniotique et le cytoplasme des cellules trophoblastiques contiennent des quantit��s anormalement ��lev��es de HMGB-1, mais il n'est toujours pas universellement admis que ces concentrations se retrouvent dans le sang maternel. M��thodes : nous avons recrut�� 32 femmes au troisi��me trimestre de grossesse, 16 avec et 16 sans pr����clampsie. Nous avons ��galement observ�� 16 femmes non enceintes et en bonne sant��, appari��es selon l'��ge avec les femmes enceintes. Nous avons mesur�� la concentration s��rique de HMGB-1 chez les femmes enceintes avant, puis 24-48 heures apr��s leur accouchement, en utilisant un kit ELISA commercial. Le m��me dosage a ��t�� r��alis�� chez les femmes non enceintes, mais �� une seule reprise, au moment de leur inclusion dans l'��tude. R��sultats : le jour de leur inclusion dans l'��tude, la concentration m��diane [intervalle interquartile] de HMGB-1 chez les femmes enceintes pr����clamptiques ��tait de 2.1 ng/ml [1.1 - 3.2], de 1.1 [1.0-1.2] chez les grossesses saines (p &lt; 0.05 vs groupe pr����clamptiques) et de 0.6 [0.5 - 0.8] chez les patientes non enceintes (p &lt; 0.01 vs deux autres groupes). Pour les deux groupes de femmes enceintes, les concentrations mesur��es en post-partum ne variaient pas significativement de celles mesur��es avant l'accouchement. Conclusion : avec ou sans pr����clampsie, le troisi��me triemstre de la grossesse est associ�� �� une ��l��vation des taux circulants de HMGB-1. Cette augmentation est exag��r��e en cas de pr����clampsie. L'origine de ces concentrations ��lev��es reste �� d��terminer, mais elle semble impliquer d'autres organes que le placenta lui-m��me.

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1�� in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1��) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.