Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

Regulation of mass and function of pancreatic β-cells: identification of anti-apoptotic peptides and role of GLP-1

Résumé La masse de cellules β sécrétrices d'insuline est un tissu dynamique qui s'adapte aux variations de la demande métabolique pour assurer une normoglycémie. Cette adaptation se fait par un changement de sécrétion d'insuline et de la masse totale des cellules β. Une perte complète ou partielle des cellules β conduit respectivement à un diabète de type 1 et de type 2. Les mécanismes qui régulent la masse de cellules β et maintiennent leur phénotype differencié sont encore peu connus. Leur identification est nécessaire pour comprendre le développement du diabète et développer des stratégies de traitement. La greffe d'îlots est une approche thérapeutique prometteuse pour le diabète de type 1, mais est limitée par une perte précoce des cellules β due à une apoptose induite par des cytokines. Afin d'améliorer la survie des cellules β lors de la greffe d'îlots, le premier but était de trouver des peptides pouvant bloquer l'apoptose induite par FasL et TNF-α. Pour ce faire, deux librairies de phages ont été criblées pour sélectionner des peptides se liant au Fas DD ou au TNFRl DD. Nous avons identifié six peptides différents. Cependant, aucun d'entre eux n'était capable de protéger les cellules de l'apoptose induite par FasL ou TNF-α. Deuxièmement, le GLP-1 est une hormone qui stimule la sécrétion d'insuline, et est impliquée dans la prolifération des cellules β, la différentiation, et inhibe l'apoptose. Nous avons fait l'hypothèse que le GLP-1 joue un rôle crucial dans le contrôle de la masse et de la fonction des cellules β. Afin de l'évaluer, une analyse par puce à ADN a été réalisée en comparant des cellules βTC-Tet traitées avec du GLP-1 à des cellules non-traitées. 376 gènes régulés ont été identifiés, dont RGS2, CREM, ICERI et DUSP14, augmentés significativement par le GLP-1. Nous avons confirmé que le GLP-1 augmente l'expression de ces gènes, aussi bien au niveau des transcripts que des protéines. De plus, nous avons montré que le GLP-1 induit leur expression par activation de la voie cAMP/PKA, et nécessite l'entrée de calcium extracellulaire. D'après leur fonction biologique, nous avons ensuite supposé que ces gènes pourraient agir comme régulateurs négatifs de la signalisation du GLP-l, et donc freiner son effet proliférateur. Pour vérifier notre hypothèse, des siRNAs contre ces gènes ont été développés, et leurs effets sur la prolifération des cellules β seront évalués ultérieurement. Abstract The pancreatic β-cell mass is a dynamic tissue which adapts to variations in metabolic demand in order to ensure normoglycemia. This adaptation occurs through a change in both insulin secretion and the total mass of ,β-cells. An absolute or relative loss of β-cells leads to type 1 and type 2 diabetes, respectively. The mechanisms that regulate the pancreatic β-cell mass and maintain the fully differentiated phenotype of the insulin-secreting β-cells are only poorly defined. Their identification is required to understand the progression of diabetes, but also to design strategies for the treatment of diabetes. Islet transplantation is a promising therapeutic approach for type 1 diabetes, but it is still limited by an early graft loss due to cytokine-induced apoptosis. In order to improve β-cell survival during islet transplantation, our first goal was to find novel blockers of FasL- and TNF-α-mediated cell death in the form of peptides. To that end, we screened two phage display libraries to select Fas DD- or TNFR1 DD-binding peptides. We identified six different small peptides. However, none of these peptides was able to prevent cells from FasL- or TNF-α-mediated apoptosis. Secondly, GLP-1 is a hormone that has been shown to stimulate insulin secretion and to be involved in β-cell proliferation, differentiation and inhibition of apoptosis. We hypothesized that GLP-1 plays a crucial role to control mass and function of β-cells. To evaluate this hypothesis, we performed a cDNA microarray analysis with GLP-1-treated βTC-Tet cells compared to untreated cells. We found 376 regulated genes, among these, RGS2, CREM, ICERI and DUSP14, which were significantly upregulated by GLP-1. We confirmed that both their mRNA and protein levels were strongly and rapidly increased after GLP-1 treatment. Moreover, we found that GLP-1 activates their expression mainly through the activation of the cAMP/PKA signaling pathway, and requires extracellular calcium entry. According to their biological function, we then hypothesized that these genes might act as negative regulators of the GLP-1 signaling. In particular, they might brake the effects of GLP-1 on β-cell proliferation. To verify this hypothesis, siRNAs against these genes were developed. The effect of these siRNAs on GLP-1-induced β-cell proliferation will be evaluated later.

Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice.

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.

Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

Unilateral ureteropelvic junction obstruction in children: long-term followup after unilateral pyeloplasty.

PURPOSE: The benefit of surgery on renal function in unilateral ureteropelvic junction stenosis (UPJS) is still debated. We evaluated renal function outcome after unilateral pyeloplasty in 53 children. MATERIALS AND METHODS: We retrospectively reviewed 123I-hippuran renography performed at diagnosis and 5 to 15 years (mean +/- SD 7 +/- 3 years) after successful pyeloplasty. UPJS was prenatally detected in 26 children because of urinary tract infection in 17 and miscellaneous reasons in 10. Relative function (RF) and absolute function were measured on background corrected renograms. Absolute function of the affected and contralateral kidneys was determined by an accumulation index (AI), representing the percent injected dose extracted by each kidney 30 to 90 seconds after the heart peak. RESULTS: Preoperatively 33 of the 53 UPJS kidneys had a decreased AI but only 8 had a RF of less than 40%, which was improved in 7 at followup. In addition, the AI improved in 29 kidneys, of which 19 (36%) normalized. Of the UPJS kidneys 14 had an initially decreased AI that remained abnormal at followup. In these kidneys preoperative RF was less than 40% in all. At followup RF was greater than 40% in 4 children, in whom the AI of the UPJS kidney did not improve but the AI of the contralateral one decreased from supranormal to normal. Seven contralateral kidneys had a supranormal AI, whereas the AI remained normal in 3, of which the RF in the UPJS kidney remained at less than 40%. The AI and RF were normal in 20 UPJS kidneys and remained normal. CONCLUSIONS: When normal, the AI and RF reflected renal function outcome similarly. The AI added relevant information in UPJS kidneys with impaired function, showing compensation of the contralateral kidney.

The effects of losartan on renal function in the newborn rabbit.

The low GFR of newborns is maintained by various factors including the renin-angiotensin system. We previously established the importance of angiotensin II in the newborn kidney, using the angiotensin-converting enzyme inhibitor perindoprilat. The present study was designed to complement these observations by evaluating the role of angiotensin-type 1 (AT(1)) receptors, using losartan, a specific AT(1)-receptor blocker. Increasing doses of losartan were infused into anesthetized, ventilated, newborn rabbits. Renal function and hemodynamic variables were assessed using inulin and para-aminohippuric acid clearances as markers of GFR and renal plasma flow, respectively. Losartan 0.1 mg/kg slightly decreased mean blood pressure (-11%) and increased diuresis (+22%). These changes can be explained by inhibition of the AT(1)-mediated vasoconstrictive and antidiuretic effects of angiotensin, and activation of vasodilating and diuretic AT(2) receptors widely expressed in the neonatal period. GFR and renal blood flow were not modified. Losartan 0.3 mg/kg decreased mean blood pressure significantly (-15%), probably by inhibiting systemic AT(1) receptors. GFR significantly decreased (-25%), whereas renal blood flow remained stable. The decrease in filtration fraction (-21%) indicates predominant efferent vasodilation. At 3 mg/kg, the systemic hypotensive effect of losartan was marked (mean blood pressure, -28%), with decreased GFR and renal blood flow (-57% and -51%, respectively), a stable filtration fraction, and an increase in renal vascular resistance by 124%. The renal response to this dose can be considered as reflex vasoconstriction of afferent and efferent arterioles, rather than specific receptor antagonism. We conclude that under physiologic conditions, the renin-angiotensin is critically involved in the maintenance of GFR in the immature kidney.

Tumor sensitizing function and mechanism of action of a RasGAP derived peptide

Cancer is the second cause of death after cardio-vascular diseases in economically developed countries. Two of the most commonly used anti-cancer therapies are chemo and radiotherapy. Despite the remarkable advances made in term of delivery and specificity of these two anti-tumor regimens, their toxicity towards healthy tissue remains a limitation. A promising approach to overcome this obstacle would be the utilization of therapeutic peptides that specifically augment the sensitivity of tumoral cells to treatments. Lower therapeutical doses would then be required to kill malignant cells, limiting toxic effects on healthy tissues. It was previously shown in our laboratory that the caspase-3 generated fragment N2 of RasGAP is able to potentiate the genotoxin-induced apoptosis selectively in cancer cells. In this work we show that fragment N2 strictly requires a cytoplasmic localization to deliver its pro-apoptotic effect in genotoxin-treated cancer cells. The tumor sensitizing capacity of fragment N2 was found to reside within the 10 amino acid sequence 317-326. Our laboratory earlier demonstrated that a peptide corresponding to amino acids 317 to 326 of RasGAP fused to the TAT cell permeable moiety, called TAT-RasGAP317.326, is able to sensitize cancer cells, but not normal cells, to genotoxin-induced apoptosis. In the present study we describe the capacity of TAT-RasGAP 317.326 to sensitize tumors to both chemo and radiotherapy in an in vivo mouse model. The molecular mechanism underlying the TAT-RasGAP 317.326-mediated sensitization starts now to be elucidated. We demonstrate that G3BP1, an endoribonuclease binding to amino acids 317-326 of RasGAP, is not involved in the sensitization mechanism. We also provide evidence showing that TAT-RasGAP3 17-326 potentiates the genotoxin-mediated activation of Bax in a tBid-dependent manner. Altogether our results show that TAT-RasGAP 317.326 could be potentially used in cancer therapy as sensitizer, in order to improve the efficacy of chemo and radiotherapy and prolong the life expectancy of cancer patients. Moreover, the understanding of the TAT-RasGAP317.326 mode of action might help to unravel the mechanisms by which cancer cells resist to chemo and radiotherapy and therefore to design more targeted and efficient anti-tumoral strategies.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

Alteration of neuromuscular function in squash

The alteration in neuromuscular function of knee extensor muscles was characterised after a squash match in 10 trained players. Maximal voluntary contraction (MVC) and surface EMG activity of vastus lateralis (VL) and vastus medialis (VM) muscles were measured before and immediately after a 1-h squash match. M-wave and twitch contractile properties were analysed following single stimuli. MVC declined (280.5+/-46.8 vs. 233.6+/-35.4 Nm, -16%; P<0.001) after the exercise and this was accompanied by an impairment of central activation, as attested by decline in voluntary activation (76.7+/-10.4 vs. 71.3+/-9.6%, -7%; P<0.05) and raw EMG activity of the two vastii (-17%; P<0.05), whereas RMS/M decrease was lesser (VL: -5%; NS and VM: -12%; P=0.10). In the fatigued state, no significant changes in M-wave amplitude (VL: -9%; VM: -5%) or duration were observed. Following exercise, the single twitch was characterised by lower peak torque (-20%; P<0.001) as well as shorter half-relaxation time (-13%; P<0.001) and reduced maximal rate of twitch tension development (-23%; P<0.001) and relaxation (-17%; P<0.05). A 1-h squash match play caused peripheral fatigue by impairing excitation-contraction coupling, whereas sarcolemmal excitability seems well preserved. Our results also emphasise the role of central activation failure as a possible mechanism contributing to the torque loss observed in knee extensors. Physical conditioners should consider these effects when defining their training programs for squash players.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Regulation of Rat and Human T-cell Immune Response by Pharmacologically Modified Dendritic Cells.

BACKGROUND: The central function of dendritic cells (DC) in inducing and preventing immune responses makes them ideal therapeutic targets for the induction of immunologic tolerance. In a rat in vivo model, we showed that dexamethasone-treated DC (Dex-DC) induced indirect pathway-mediated regulation and that CD4+CD25+ T cells were involved in the observed effects. The aim of the present study was to investigate the mechanisms underlying the acquired immunoregulatory properties of Dex-DC in the rat and human experimental systems. METHODS: After treatment with dexamethasone (Dex), the immunogenicity of Dex-DC was analyzed in T-cell proliferation and two-step hyporesponsiveness induction assays. After carboxyfluorescein diacetate succinimidyl ester labeling, CD4+CD25+ regulatory T-cell expansion was analyzed by flow cytometry, and cytokine secretion was measured by ELISA. RESULTS: In this study, we demonstrate in vitro that rat Dex-DC induced selective expansion of CD4+CD25+ regulatory T cells, which were responsible for alloantigen-specific hyporesponsiveness. The induction of regulatory T-cell division by rat Dex-DC was due to secretion of interleukin (IL)-2 by DC. Similarly, in human studies, monocyte-derived Dex-DC were also poorly immunogenic, were able to induce T-cell anergy in vitro, and expand a population of T cells with regulatory functions. This was accompanied by a change in the cytokine profile in DC and T cells in favor of IL-10. CONCLUSION: These data suggest that Dex-DC induced tolerance by different mechanisms in the two systems studied. Both rat and human Dex-DC were able to induce and expand regulatory T cells, which occurred in an IL-2 dependent manner in the rat system.

Capsid modified replicating adenovirus to selectively target colon cancer

Résumé: Pratiquement tous les cancers du colon contiennent des mutations dans la voie de signalisation de Wnt qui active constitutivement cette voie. Cette activation mène à la stabilisation de la β-catenine. La β-catenin est transportée dans le noyau ou elle active des gènes cible en interagissant avec le facteur de transcription de TCF/LEF. Des adénovirus qui peuvent sélectivement se répliquer dans les cellules tumorales sont les agents qui peuvent permettre la déstruction de la tumeur mais pas le tissu normal. In vitro, les adénovirus avec des sites d'attachement du facteur de transcription TCF dans les promoteurs de l'adénovirus montrent une sélectivité et une activité dans une large sélection de lignées cellulaires de cancer du colon. Au contraire, in vivo, quand les adénovirus modifiés sont injectés dans la circulation, ils sont moins efficaces à cause de leur fixation par le foie et à cause de l'absence d'expression du récepteur du Coxsackie-Adénovirus (CAR). Le but de ma thèse était de modifier la protéine principale de capside de l'adénovirus, fibre, pour augmenter l'infection des tumeurs du cancer du colon. La fibre de l'adénovirus est responsable de l'attachement aux cellules et de l'entrée virale. J'ai inséré un peptide RGD dans la boucle HI de la fibre qui dirige sélectivement le virus aux récepteurs des integrines. Les integrines sont surexprimées par les cellules du cancer du colon et l'endothélium des vesseaux de la tumeur. Le virus re-ciblé, vKH6, a montré une activité accrue dans toutes les lignées cellulaires de cancer du colon, tandis que la sélectivité était maintenue. In vivo, vKH6 était supérieur au virus avec une capside de type sauvage en retardant la croissance de la tumeur. Le virus s'est répliqué plus vite et dispersé graduellement dans la tumeur. Cet effet a été montré par hybridation in situ et par PCR quantitative. Cependant, la monothérapie avec le virus n'a pu retarder la croissance des cellules tumorales SW620 greffées que de 2 semaines, mais à cause des régions non infectées la tumeur n'a pas pu être éliminée. Bien que la combinaison avec les chimiothérapies conventionnelles soit d'intérêt potentiel, presque toutes interfèrent avec la réplication virale. Les drogues antiangiogéniques sont des agents anti-tumoraux efficaces et prometteurs. Ces drogues n'interfèrent pas avec le cycle de vie de l'adénovirus. RAD001 est un dérivé de la rapamycine et il inhibe mTOR, une protéine kinase de la voie de PI3K. RAD001 empêche la croissance des cellules et il a aussi des effets anti-angiogénique et immunosuppressifs. RAD001 in vitro n'affecte pas l'expression des gènes viraux et la production virale. La combinaison de VKH6 et RAD001 in vivo a un effet additif en retardant la croissance de la tumeur. Des nouveaux peptides plus efficaces dans le ciblage de l'adénovirus sont nécessaires pour augmenter l'infection des tumeurs. J'ai créé un système de recombinaison qui permettra la sélection de nouveaux peptides dans le contexte du génome de l'adénovirus. Summary Virtually all colon cancers have mutations in the Wnt signalling pathway which result in the constitutive activation of the pathway. This activation leads to stabilization of β-catenin. β-catenin enters the nucleus and activates its target genes through interaction with the TCF transcription factor. Selectively replicating adenoviruses are promising novel agents that can destroy the tumour but not the surrounding normal tissue. In vitro, adenoviruses with TCF binding sites in the early viral promoters show selectivity and activity in a broad panel of viruses but in vivo they are less effective due to the lack of expression of the Coxsackie-Adenovirus receptor (CAR). The aim of my thesis was to modify the major capsid protein of the adenovirus, fibre, to increase the infection of colon tumours. Fibre of adenovirus is responsible for the binding to cells and for the viral uptake. I inserted an RGD binding peptide into the HI loop of fibre that selectively targets the virus to integrins that are overexpressed on tumour cells and on tumour endothelium. The retargeted virus, vKH6, showed increased activity in all colon cancer cell lines while selectivity was maintained. In vivo, vKH6 is superior to a matched virus with a wild type capsid in delaying tumour growth. vKH6 replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. The virus alone can delay the growth of SW620 xenografts by 2 weeks but due to uninfected tumour regions the tumour cannot be cured. Although combination with conventional chemotherapeutics is of potential interest, almost all of them interfere with the viral replication. Growing evidence supports that anti-angiogenic drugs are effective and promising anti-tumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it blocks mTOR, a protein kinase in the PI3K pathway. RAD001 inhibits cell growth and has strong anti-angiogenic and immunosuppressive effects. RAD001 in vitro does not affect viral gene expression and viral burst size. In vivo vKH6 and RAD001 have an additive effect in delaying tumour growth, but tumour growth is still not completely inhibited. To further increase tumour infection new tumour specific targeting peptides are needed. I created an adenovirus display library that will allow the selection of targeting peptides. This system may also facilitate the production of fibre modified viruses.

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice.

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1(-/-) mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1(-/-) mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1(-/-) mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1(-/-) mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload.

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Catechol-O-methyltransferase, dopamine, and sleep-wake regulation.

NlmCategory="UNASSIGNED">Sleep and sleep disorders are complex and highly variable phenotypes regulated by many genes and environment. The catechol-O-methyltransferase (COMT) gene is an interesting candidate, being one of the major mammalian enzymes involved in the catabolism of catecholamines. The activity of COMT enzyme is genetically polymorphic due to a guanine-to-adenine transition at codon 158, resulting in a valine (Val) to methionine (Met) substitution. Individuals homozygous for the Val allele show higher COMT activity, and lower dopaminergic signaling in prefrontal cortex (PFC) than subjects homozygous for the Met allele. Since COMT has a crucial role in metabolising dopamine, it was suggested that the common functional polymorphism in the COMT gene impacts on cognitive function related to PFC, sleep-wake regulation, and potentially on sleep pathologies. The COMT Val158Met polymorphism may predict inter-individual differences in brain electroencephalography (EEG) alpha oscillations and recovery processes resulting from partial sleep loss in healthy individuals. The Val158Met polymorphism also exerts a sexual dimorphism and has a strong effect on objective daytime sleepiness in patients with narcolepsy-cataplexy. Since the COMT enzyme inactivates catecholamines, it was hypothesized that the response to stimulant drugs differs between COMT genotypes. Modafinil maintained executive functioning performance and vigilant attention throughout sleep deprivation in subjects with Val/Val genotype, but less in those with Met/Met genotype. Also, homozygous Met/Met patients with narcolepsy responded to lower doses of modafinil compared to Val/Val carriers. We review here the critical role of the common functional COMT gene polymorphism, COMT enzyme activity, and the prefrontal dopamine levels in the regulation of sleep and wakefulness in normal subjects, in narcolepsy and other sleep-related disorders, and its impact on the response to psychostimulants.