Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Investigation of potential intervention targets to improve insulin action: a therapeutic approach

Impaired insulin action (insulin resistance) is a key factor in the pathogenesis of diabetes mellitus. To investigate therapeutic targets against insulin resistance, this thesis explores the mechanism of action of pharmacological agents and exogenous peptides known or suspected to modify insulin action. These included leptin, a hormone primarily involved in the regulation of body weight; sibutramine, an antiobesity agent; plant-derived compounds (pinitol and chamaemeloside) and agents known to affect insulin sensitivity, e.g. metformin, tolbutamide, thiazolidinediones, vanadyl sulphate and thioctic acid. Models used for investigation included the L6 skeletal muscle cell line and isolated skeletal muscles. In vivo studies were undertaken to investigate glycaemia, insulinaemia, satiety and body weight in streptozotocin-induced diabetic mice and obese (ob/ob) mice. Leptin acutely altered insulin action in skeletal muscle cells via the short form of the leptin receptor. This direct action of leptin was mediated via a pathway involving PI 3-kinase but not Jak2. The active metabolites of sibutramine had antidiabetic properties in vivo and directly improved insulin sensitivity in vitro. This effect appeared to be conducted via a non-PI 3-kinase-mediated increase in protein synthesis with facilitated glucose transport, and was independent of the serotonin and noradrenaline reuptake inhibition produced by sibutramine. Pinitol (a methyl inositol) had an insulin mimetic effect and was an effective glucose-lowering agent in insulinopenic states, acting directly on skeletal muscle. Conversely chamaemeloside appeared to improve glucose tolerance without directly altering glucose transport. Metformin directly increased basal glucose uptake independently of PI 3-kinase, possibly via an increase in the intrinsic activity of glucose transporters. Neither tolbutamide nor thiazolidinediones directly altered insulin sensitivity in L6 skeletal muscle cells: however vanadyl sulphate and thioctic acid increased glucose transport but appeared to exert toxic effects at therapeutic concentrations. Examination of glucose transport in skeletal muscle in this thesis has identified various components of post-receptor insulin signalling pathways which may be targeted to ameliorate insulin resistance. Type 2 Diabetes Mellitus Obesity L6 Skeletal Muscle Cells Glucose Transport Insulin Signalling 2

Antibacterial activity of low amperage and low voltage electric current (DC)

Infection is a major clinical problem associated with the use of intravenous catheters.The efficacy of a direct electric current (10µA, 9V) via electrode-conducting carbon impregnated catheters to prevent colonisation of catheters by micro-organisms was investigated. The range of organisms susceptible to 10µA was determined by a zone of inhibition test. The catheters acting as the anode and the cathode were inserted into a nutrient agar plate inoculated with a lawn of bacteria. There was no zone of inhibition observed around the anode. Organisms susceptible to 10µA at the cathode were Staphylococcus aureus (2 strains), Staphylococcus epidermidis (5 strains), Escherichia coli and Klebsiella pneumoniae (2 strains each), and one strain of the following micro-organisms: Staphylococcus hominis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The zones ranged from 6 to 16 mm in diameter according to the organisms under test. The zone size was proportional to the amperage (10 - 100 µA) and the number of organisms on the plate. Ten µA did not prevent adhesion of staphylococci to the cathode nor did it affect their growth in nutrient broth. However, it was bactericidal to adherent bacteria on the cathodal catheter and significantly reduced the number of bacteria on the catheter after 4 to 24 h application of electricity. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated.The mechanisms of the bactericidal activity associated with the cathode were investigated with S. epidermidis and S. aureus. The inhibition zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. Hydrogen peroxide was produced at the cathode surface under aerobic conditions, but not in the absence of oxygen. A salt-bridge apparatus was used to demonstrate further that hydrogen peroxide was produced at the cathode, and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of hydrogen peroxide as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting chlorine plays only a minor role in the bactericidal activity against organisms attached to anodal electrode surfaces. The bactericidal activity of electric current associated with the cathode and H202 was greatly reduced in the presence of 50 μM to 0.5 mM magnesium ions in the test menstrum. Ten μA applied via the catheters did not prevent the initial biofilm growth by the adherent bacteria but reduced the number of bacteria in the biofilm by 2 log order aiter 24 h. The results suggested that 10 μA may prevent the colonisation of catheters by both the extra~ and intra-luminal routes. The localised production of hydrogen peroxide and chlorine and the intrinsic activity due to electric current may offer a useful method for the eradication of bacteria from catheter surfaces.

Theoretical Study of Heteroatom Doping in Tuning the Catalytic Activity of Graphene for Triiodide Reduction

Graphene with heteroatom doping has found increasing applications in a broad range of catalytic reactions. However, the doping effects accounting for the enhanced catalytic activity still remain elusive. In this work, taking the triiodide electroreduction reaction as an example, we study systematically the intrinsic activity of graphene and explore the origin of doping-induced activity variation using first-principles calculations, in which two typical N and S dopants are tested. The most common graphene structures, basal plane, armchair edge, and zigzag edge, are considered, and it is found that the former two structures show a weak adsorption ability for the iodine atom (the key intermediate in the triiodide electroreduction reaction), corresponding to a low catalytic activity. Doping either N or S can strengthen the adsorption and thus increase the activity, and the codoping of N and S (NS-G) exhibits a synergistic effect. A detailed investigation into the whole process of the triiodide electroreduction reaction at the CH3CN/NS-G interface is also carried out to verify these activity trends. It is found that the zigzag edges which contain spin electrons show a relatively stronger adsorption strength compared with the basal plane and armchair edge, and initial doping would result in the spin disappearance that evidently weakens the adsorption; with the disappearance of spin, however, further doping can increase the adsorption again, suggesting that the spin electrons may play a preliminary role in affecting the intrinsic activity of graphene. We also analyzed extensively the origin of doping-induced adsorption enhancement of graphene in the absence of spin; it can be rationalized from the electronic and geometric factors. Specifically, N doping can result in a more delocalized “electron-donating area” to enhance I adsorption, while S doping provides a localized structural distortion, which activates the nearest sp2-C into coordinatively unsaturated sp3-C. These results explain well the improved activity of the doping and the synergistic effect of the codoping. The understandings are generalized to provide insight into the enhanced activity of the oxygen reduction reaction on heteroatom doped graphene. This work may be of importance toward the design of high-activity graphene based material.

EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Determination of the solution structures of conantokin-G and conantokin-T by CD and NMR spectroscopy

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses, They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational gamma-carboxyglutamic acid (Gla) residues, Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form alpha-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold, We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and H-1 NMR spec troscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from H-1 NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of alpha- and 3(10) helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, Or Mg2+) to form a stable iv-helix, while conantokin-T adopts a stable alpha-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, Or Mg2+).

Catálise de hydrocraking baseado em zeólitos

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química

Differences of susceptibility of five triatomine species to pyrethroid insecticides - implications for Chagas disease vector control

As pyrethroids are presently the favored group of insecticides to control triatomines, we performed a series of bioassays to determine the intrinsic activity of some of the main compounds used in the control campaigns, against five of the main species of triatomines to be controlled. Comparing the insecticides it can be seen that lambdacyhalothrin is more effective than the other three pyrethroids, both considering the LD50 and 99 for all the three species with comparable results. On Triatoma infestans the LD50 of lambdacyhalothrin was followed by that of alfacypermethrin, cyfluthrin and deltamethrin. On Rhodnius prolixus the sequence, in decreasing order of activity, was lambdacyhalothrin, alfacypermethrin, deltamethrin and cyfluthrin. Some modifications can be seen when we compare the LD99, that has more to see to what happens in the field. T. brasiliensis showed to be as sensible to lambdacyhalothrin as T. infestans, the most susceptible for this product. By the other side T. sordida is the least susceptible considering the LD99 of this insecticide.

Enforced covalent trimerisation of soluble feline CD134 (OX40)-ligand generates a functional antagonist of feline immunodeficiency virus.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumour necrosis factor receptor superfamily (TNFRSF) and all primary viral strains tested to date use CD134 for infection. To investigate the effect of the natural ligand for CD134 on FIV infection, feline CD134L was cloned and expressed in soluble forms. However, in contrast to murine or human CD134L, soluble feline CD134L (sCD134L) did not bind to CD134. Receptor-binding activity was restored by enforced covalent trimerisation following the introduction of a synthetic trimerisation domain from tenascin (TNC). Feline and human TNC-CD134Ls retained the species-specificity of the membrane-bound forms of the ligand while murine TNC-CD134L displayed promiscuous binding to feline, human or murine CD134. Feline and murine TNC-CD134Ls were antagonists of FIV infection; however, potency was both strain-specific and substrate-dependent, indicating that the modulatory effects of endogenous sCD134L, or exogenous CD134Lbased therapeutics, may vary depending on the viral strain.

Analyse fonctionnelle et moléculaire d'un nouveau gène (VIT32) impliqué dans le mécanisme d'action de la vasopressine

Résumé Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale (tube distal et tube collecteur) du néphron ont un rôle essentiel dans le maintien de l'homéostasie de la composition ionique et du volume des fluides extracellulaires. Ces mécanismes permettent le maintien de la pression sanguine. Dans la cellule principale du tube collecteur cortical (CCD), le taux de réabsorption de sodium dépend essentiellement de l'activité du canal épithélial à sodium (ENaC) à la membrane apicale et de la pompe sodium-potassium-adénosine-triphosphatase (Na+-K±ATPase) à la membrane basolatérale. L'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales de CCD, la vasopressine régule le transport de sodium en deux étapes : une étape précoce dite « non-génomique » et une étape tardive dite « génotnique ». Durant l'étape précoce, la vasopressine régule l'expression de gènes, dont certains peuvent être impliqués dans le transport de sodium, comme ENaC et la Na+ -K+ATP ase. Le but de mon travail a été d'étudier l'implication d'une protéine appelée VIP32 (vasopressin induced protein : VIP) dans le transport de sodium. L'expression de VIP32 est augmentée par la vasopressine dans les cellules principales de CCD. Dans l'ovocyte de Xenopus laevis utilisé comme système d'expression hétérologue, nous avons montré que l'expression de VIP32 provoque la maturation méiotique de l'ovocyte par l'activation de la voie des MAPK (mitogen-activated protein kinase : MAPK) et du facteur de promotion méiotique (MPF). La co-expression d'ENaC et de VIP32 diminue l'activité d'ENaC de façon sélective, par l'activation de la voie des MAPK, sans affecter l'expression du canal à la surface membranaire. Nous avons également montré que la régulation de l'activité d'ENaC par la voie des MAPK est dépendante du mécanisme de régulation d'ENaC lié à un motif du canal appelé PY. Ce motif est impliqué dans le contrôle de la probabilité d'ouverture ainsi que l'expression à la surface membranaire d'ENaC. Dans les cellules principales, VIP32 par l'activation de la voie des MAPK peut être impliqué dans la régulation négative du transport transépithélial qui a lieu après plusieurs heures de traitement à la vasopressine. Le tube collecteur de reins normaux présente un taux basal significatif d'activité de la voie MAPK MEK1/2-ERK1/2. Dans la lignée mpkCCDc14 de cellules principales de CCD de souris, que nous avons utilisé pour cette partie du travail, nous avons montré la présence d'un taux basal d'activité d'ERK1/2 (pERK1/2). L'aldostérone et la vasopressine, connus pour stimuler le courant sodique transépithélial dans le CCD, ne changeaient pas le taux basal de pERK1/2. Le transport de sodium transépithélial basal, ou stimulé par l'aldostérone ou la vasopressine est diminué par l'effet de PD98059, un inhibiteur de MEK1/2 qui diminue parallèlement le taux de pERK1/2. Nous avons également montré dans des cellules non stimulées, ou stimulées par de l'aldostérone ou de la vasopressine, que l'activité de la Na+-K+ ATPase, mais pas celle d'ENaC est inhibée par des traitements avec différents inhibiteurs de MEK1/2. Par un marquage de la Na±-K+ATPase à la surface membranaire nous avons montré que la voie d'ERK1/2 contrôle l'activité intrinsèque de la Na+-K+ ATPase, plutôt que son expression à la surface membranaire. Ces données ont montré que l'activité de la Na+-K+ATPase et le transport transépithélial de sodium sont contrôlés par l'activité basal et constitutive de la voie d'ERK1/2. Summary The regulation of sodium reabsorption in the distal nephron (distal tubule and cortical collecting duct) in the kidney plays an essential role in the control of extracellular fluids composition and volume, and thereby blood pressure. In the principal cell of the collecting duct (CCD), the level of sodium reabsorption mainlly depends on the activity of both epithlial sodium channel (ENaC) and sodium-potassium-adenosine-triphosphatase (Na+-K+ATPase). The activity of these two transporters is regulated by hormones especially aldosterone, vasopressin and insuline.In the principal cell of the CCD, vasopressin regulates sodium transport via a short-term effect and a late genomic effect. During the genomic effect vasopressin activates a complex network of vasopressin-dependent genes involved in the regulation of sodium transport as ENaC and Na+-K+ATPase. We were interested in the role of a recently identified vasopressin induced protein (VIP32) and its implication in the regulation of sodium transport in principal cell of the CCD. The Xenopus oocyte expression system revealed two functions : expressed alone VIP32 rapidly induces oocyte meiotic maturation through the activation of the mitogen-activated protein kinase (MAPK) pathway and the meiotic promoting factor and when co-expressed with ENaC, V1P32 selectively dowrn-egulates channel activity, but not channel cell surface expression. We have shown that the ENaC downregulation mediated by the activation of the MAPK pathway is related to the PY motif of ENaC. This motif is implicated in ENaC cell surface expression and open probability regulation. In the kidney principal cell, VIP32 through the activation of MAPK pathway may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 MAPK pathway. Using in vitro cultured mpkCCDc14 principal cells we have shown a significant basal level of ERK1/2 activity (pERK1/2). Aldosterone and vasopressin, known to upregulate sodium reabsorption in CCDs, did not change ERK1/2 activity. Basal and aldosterone- or vasopressin-stimulated sodium transport were downregulated by the MEK1/2 inhibitor PD98059 in parallel with a decrease in pERK1/2 in vitro. The activity of Na+-K+ATPase but not that of ENaC was inhibited by MEK1/2 inhibitors in both, unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface labelling showed that intrinsic activity rather than cell surface expression of Na+-K+ATPase was controlled by pERK1/2. Our data demonstrate that basal constitutive activity of ERK1/2 pathway controls Na+-K+ATPase activity and transepithelial sodium transport in the principal cell. Résumé tout public Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale du néphron (l'unité fonctionnelle du rein) ont un rôle essentiel dans le maintien de l'homéostasie de la composition et du volume des fluides extracellulaires. Ces mécanismes permettent de maintenir une pression sanguine effective. Dans les cellules principales du tube collecteur, une région spécifique du néphron distal, le transport de sodium dépend essentiellement de l'activité de deux transporteurs de sodium : le canal épithélial à sodium (ENaC) et la pompe sodium-potassium-adénosine-triphosphatase (Na+-K+ATPase). Afin de répondre aux besoins de l'organisme, l'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales du tube collecteur, la vasopressine régule le transport de sodium en deux étapes : une étape rapide et une étape lente dite « génomique ». Durant l'étape lente, la vasopressine régule l'expression de gènes pouvant être impliqués dans le transport de sodium, dont notamment ceux d'ENaC et de la Na+-K+ATPase. Parmi les gènes dont l'expression est augmentée par la vasopressine, celui de VIP32 (vasopressin induced protein : VIP) fait l'objet de cette étude. Le but de mon travail a été d'étudier, dans un système d'expression hétérologue (l'ovocyte de Xenopus leavis), l'implication de VIP32 dans le transport de sodium. Nous avons montré que VIP32 est capable d'activer un mécanisme moléculaire en cascade appelé MAPK (mitogen-activated protein kinase : MAPK) et est aussi capable de diminuer l'activité d'ENaC. Parallèlement, dans une lignée de cellules principales de tube collecteur les mpkCCDc14, nous avons montré que le taux basal d'activité de la cascade MAPK est capable de réguler l'activité de la Na+-K+ATPase, tandis qu'il n'influence pas l'activité d'ENaC.

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol).CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Absence of peripheral blood mononuclear cells priming in hemodialysis patients

As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC) priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP) and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6) cells). Lipopolysaccharide (5 µg/ml) did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.

Rôle du GPR91 dans la réponse à l'hypoxie-ischémie et l'importance de sa localisation intracellulaire

L'adaptation à l'environnement est essentielle à la survie cellulaire et des organismes en général. La capacité d'adaptation aux variations en oxygène repose sur des mécanismes de détection de l'hypoxie et une capacité à répondre en amorçant un programme d'angiogenèse. Bien que la contribution du facteur induit par l'hypoxie (HIF) est bien définie dans l'induction d'une telle réponse, d'autres mécanismes sont susceptibles d'être impliqués. Dans cette optique, les études démontrant l'influence du métabolisme énergétique sur le développement vasculaire sont de plus en plus nombreuses. L'un de ces composés, le succinate, a récemment été démontré comme étant le ligand du GPR91, un récepteur couplé aux protéines G. Parmi les différents rôles attribués à ce récepteur, notre laboratoire s'intéressa aux rôles du GPR91 dans la revascularisation observée suite à des situations d'hypoxie dont ceux affectant la rétine. Il existe cependant d'autres conditions pour lesquelles une revascularisation serait bénéfique notamment suite à un stress hypoxique-ischémique cérébral. Nos travaux ont pour objectifs de mieux comprendre le rôle et le fonctionnement de ce récepteur durant le développement et dans le cadre de pathologies affectant la formation de vaisseaux sanguins. Dans un premier temps, nous avons déterminé le rôle du GPR91 dans la guérison suite à un stress hypoxique-ischémique cérébral chez le nouveau-né. Nous montrons que ce récepteur est exprimé dans le cerveau et en utilisant des souris n'exprimant pas le GPR91, nous démontrons que dans un modèle d'hypoxie-ischémie cérébrale néonatal l'angiogenèse prenant place au cours de la phase de guérison dépend largement du récepteur. L'injection intracérébrale de succinate induit également l'expression de nombreux facteurs proangiogéniques et les résultats suggèrent que le GPR91 contrôle la production de ces facteurs. De plus, l'injection de ce métabolite avant le modèle d'hypoxie-ischémie réduit substantiellement la taille de l'infarctus. In vitro, des essaies de transcription génique démontrent qu'à la fois les neurones et les astrocytes répondent au succinate en induisant l'expression de facteurs bénéfiques à la revascularisation. En considérant le rôle physiologique important du GPR91, une seconde étude a été entreprise afin de comprendre les déterminants moléculaires régissant son activité. Bien que la localisation subcellulaire des RCPG ait traditionnellement été considérée comme étant la membrane plasmique, un nombre de publications indique la présence de ces récepteurs à l'intérieur de la cellule. En effet, tel qu'observé par microscopie confocale, le récepteur colocalise avec plusieurs marqueurs du réticulum endoplasmique, que celui-ci soit exprimé de façon endogène ou transfecté transitoirement. De plus, l’activation des gènes par stimulation avec le succinate est fortement affectée en présence d'inhibiteur du transport d'acides organiques. Nous montrons que le profil de facteurs angiogéniques est influencé selon la localisation ce qui affecte directement l'organisation du réseau tubulaire ex vivo. Finalement, nous avons identifié une région conservée du GPR91 qui agit de signal de rétention. De plus, nous avons découvert l'effet de l'hypoxie sur la localisation. Ces travaux confirment le rôle de régulateur maître de l'angiogenèse du GPR91 lors d'accumulation de succinate en condition hypoxique et démontrent pour la première fois l'existence, et l'importance, d'un récepteur intracellulaire activé par un intermédiaire du métabolisme. Ces données pavent donc la voie à une nouvelle avenue de traitement ciblant GPR91 dans des pathologies hypoxiques ischémiques cérébrales et soulèvent l'importance de tenir compte de la localisation subcellulaire de la cible dans le processus de découverte du médicament.