Role of amphiregulin in mammary gland development

Abstract Ovarian hormones are key regulators of postnatal mammary gland development and are linked to breast carcinogenesis. In particular, estrogens induce mammary epithelial cells to proliferate at the onset of puberty, leading to the elongation of the rudimental ductal tree into the fatty stromal tissue. Elucidating the molecular events underlying estrogen mitogenic activity in the mammary gland is of value in understanding how the deregulation of this signalling pathway can lead to breast tumorigenesis. Our lab has recently shown that estrogen induces mammary proliferation via epithelial estrogen receptor alpha (ERα) by a paracrine mechanism. Based on the finding that several EGF receptor (EGFR) ligands are able to substitute for estrogens and that amphiregulin (Areg), one of these ligands, is required during mammary development, we have hypothesized that Areg is a key mediator of estrogen induced paracrine signalling during ductal morphogenesis. Our analysis of the Areg -/- mice mammary phenotype reveals that epithelial Areg is required at the onset of puberty for epithelial proliferation, terminal end bud (TEB) formation and, subsequently, ductal elongation. Hormonal stimulation experiments show that among the EGFR ligands, only Arég is specifically controlled by estrogen at the transcriptional level, via ERα, in the mammary gland. Moreover, Areg is required for the estrogen-induced mammotrophic effects of epithelial proliferation and ductal elongation. We have shown that ectopic Areg expression in ERα -/- mammary epithelial cells is sufficient to induce ductal morphogenesis. Our transplantations experiment show that when Areg -/- cells are in the presence of wt cells they contribute to all aspects of ductal development, suggesting that this growth factor acts in a paracrine fashion. Moreover, this result shows that Areg -/- epithelial cells are not intrinsically impaired in proliferation. Our transplantation experiment carried out under physiological conditions confirmed previous reports showing that stromal EGFR is needed for ductal morphogenesis. This suggests that estrogen-driven Areg signalling involves an epithelium-stroma crosstalk. Thus, these data confirmed our hypothesis of Areg being an important estrogen mediator during ductal morphogenesis. Résumé Les hormones ovariennes, régulatrices clés du développement post-natal de la glande mammaire, sont également liées à la carcinogénèse du sein. En particulier, l'oestrogène induit la division des cellules épithéliales au début de la puberté. Cette prolifération amène à l'élongation du réseau canalaire rudimentaire et permet l'invasion du compartiment stromal. L'élucidation des mécanismes moléculaires responsables de l'activité mitogénique de l'oestrogène dans la glande mammaire est précieuse pour une meilleure compréhension du développement du cancer du sein. Notre laboratoire a récemment démontré que l'cestrogène induit la prolifération des cellules épithéliales par un signal paracrine, grâce au récepteur à l'oestrogène alpha (ERα). En se basant sur le fait que plusieurs ligands du récepteur à l'EGF (EGFR) sont capables de se substituer à l'cestrogène et d'induire la prolifération épithéliale et qu'amphiregulin (Areg), un de ces ligands, est essentielle au développement de la glande mammaire, nous avons émi l'hypothèse que Areg est un médiateur essentiel du signal paracrine induit par l'oestrogène pendant la croissance du système canalaire. Nos analyses phénotypiques des glandes mammaires issues de souris transgéniques Areg -/- démontrent que cette protéine est indispensable à la prolifération des cellules épithéliales mammaires au début de la puberté et à la formation des bourgeons terminaux qui conduisent à l'élongation des canaux. Nos expériences de stimulations hormonales démontrent que, parmi l'ensemble des ligands du EGFR, seule Areg est contrôlée au niveau transcriptionnel par l'cestrogène dans la glande mammaire, ceci via le récepteur ERα. De plus, Areg est essentielle pour le effets mammotrophique induit par l'cestrogène, à savoir la prolifération épithéliál et la croissance du système canalaire. Par ailleurs, l'expression ectopique d'Areg dans des cellules epithéliales mammaires de souris transgéniques ERα -/- est suffisante pour permettre la formation du réseau canalaire. En présence de cellules normales, les cellules dépourvues du gène d'Areg contribuent à la formation des canaux. Cette expérience suggère que ce facteur de croissance agit de manière paracrine. De plus, ce résultat montre que les cellules épithéliales Areg -/- conservent leur potentiel prolifératif. Nos expériences de transplantation, réalisées dans des conditions physiologiques, ont confirmé des précédentes études qui montraient que le récepteur stromal à l'EGF est nécessaire pour la morphogénèse du système canalaire. Ceci suggère que la voie de signalisation activée par l'oestrogène et dépendante d' implique une communication entre l'épithélium et le stroma. Ainsi, ces résultats valident notre hypothèse puisqu'ils confirment Areg en tant que médiateur majeur de l'oestrogène dans la morphogénèse du système canalaire.

KIAA1985, a Protein Mutant in Charcot-Marie-Tooth Neuropathy, Links Peripheral Nerve Myelination to Endosomal Recycling Pathways

Charcot-Marie-Tooth neuropathy (CMT) represents a heterogenous group of inherited disorders of the peripheral nervous system. One form of autosomal recessive demyelinating CMT (CMT4C, 5q32) is caused by mutations in the gene encoding KIAA1985, a protein of so far unknown function. Here we show that KIAA1985 is exclusively expressed in Schwann cells. KIAA1985 is tethered to cellular membranes through an N-terminal myristic acid anchor and localizes to the perinuclear recycling compartment. A search for proteins that interact with KIAA1985 identified the small GTPase Rab11, a key regulator of recycling endosome functions. CMT4C-related missense mutations disrupt the KIAA1985/Rab11 interaction. Protein binding studies indicate that KIAA1985 functions as a Rab11 effector, as it interacts only with active forms of Rab11 (WT and Q70L) and does not interact with the GDP locked mutant (S25N). Consistent with a function of Rab11 in Schwann cell myelination, myelin formation was strongly impaired when dorsal root ganglion neurons were co-cultured with Schwann cells infected with Rab11 S25N. Our data indicate that the KIAA1985/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

Study of the physiological role of RasGAP cleavage

Résume Les caspases sont un groupe de protéases à cystéine qui s?activent lors de l'apoptose. Leur activation induit le clivage de nombreuses cibles intracellulaires, conduisant à l'activation de voies pro-apoptotiques et finalement au démantèlement des cellules. Cependant, des caspases ont été décrites dans de nombreux autres processus indépendants de l'apoptose, notamment dans la physiologie des cellules hématopoïétiques, des cellules musculaires, des cellules de la peau et des neurones. Comment est-ce que les cellules réconcilient-elles ces deux fonctions distinctes? Une partie de la réponse réside dans la nature des substrats qu'elles clivent. Certains substrats, une fois clivées, deviennent anti-apoptotiques. RasGAP est une cible des caspases et contient deux sites spécifiques de clivage par les caspases. Lorsque le niveau d?activité des caspases est faible le clivage de RasGAP produit un fragment N-terminal qui active un signal antiapoptotique, relayé par la voie de Ras/PI3K/Akt. Lorsque le niveau d?activité des caspases est plus élevé le fragment RasGAP N-terminal est à nouveau clivé, perdant de ce fait ses propriétés anti-apoptotiques. Dans cette étude, nous avons mis en évidence que l'activation de la voie Ras/PI3K/Akt induite par le fragment RasGAP N-terminal dépend de RasGAP lui-même. Par ailleurs, dans le but d?étudier l?importance du clivage de RasGAP dans un contexte physiologique, nous avons développé un modèle animal exprimant une gêne mutée de RasGAP de sorte que la protéine est devenu insensible a l?action de caspases. Les données préliminaires obtenues montrent que le clivage de RasGAP n'est pas indispensable pour le développement et l?homéostasie chez la souris. Finalement, nous avons développé une souris transgénique surexprimant le fragment de RasGAP N-terminal dans les cellules ß du pancréas. Les animaux obtenus ne montrent pas de symptômes dans les conditions basales bien qu?ils soient plus résistants au diabète induit expérimentalement. Ces résultats montrent que la surexpression du fragment N-terminal de RasGAP protége efficacement les cellules ß du pancréas de l?apoptose induite par le stress sans pourtant affecter d?autres paramètres physiologiques des Ilot de Langerhans.<br/><br/>Caspases are a series of proteases that are activated during apoptosis. Their activation causes the cleavage of numerous intracellular targets, which leads to cell dismantling and activation of pro-apoptotic pathways. Caspases have been found to be involved in the physiology of numerous cell types including haematopoietic cells, muscle cells, skin cells and neurons. How cells conciliate these two opposite functions? Part of the answer lies in the nature of the substrates they cleave. Some substrates become anti-apoptotic once cleaved by caspases. RasGAP is a caspase substrate that possesses two conserved caspase-cleavage sites. At low caspase activity, RasGAP is first cleaved and the generated N-terminal fragment activates a potent anti-apoptotic signal, mediated by the Ras/PI3K/Akt pathway. At higher caspase activity, the N-terminal fragment is further cleaved thereby losing its anti-apoptotic properties. In the present study we show that the activation of the Ras/PI3K/Akt pathway mediated by RasGAP N-terminal fragment is dependent on RasGAP itself. Moreover, to study the role of RasGAP cleavage in a physiological model, we have developed a knock-in mouse model expressing a RasGAP mutant that is not cleavable by caspases. Preliminary data shows that RasGAP cleavage is not required for normal development and homeostasis in mice. Finally, we have developed a transgenic mouse model overexpressing RasGAP N-terminal fragment in the ß-cell of the pancreas. In basal conditions, these mice show no difference with their wt counterparts. However, they are protected against experimentally induced diabetes. These results indicate that fragment N can protect ? cells from stress-induced apoptosis without affecting other physiological parameters of the Islets.

Colonic perianastomotic carcinogenesis in an experimental model

Background To examine the effect of anastomosis on experimental carcinogenesis in the colon of rats. Methods Forty-three 10-week-old male and female Sprague-Dawley rats were operated on by performing an end-to-side ileorectostomy. Group A:16 rats received no treatment. Group B: 27 rats received 18 subcutaneous injections weekly at a dose of 21 mg/kg wt of 1–2 dimethylhydrazine (DMH), from the eighth day after the intervention. Animals were sacrificed between 25–27 weeks. The number of tumours, their localization, size and microscopic characteristics were recorded. A paired chi-squared analysis was performed comparing tumoral induction in the perianastomotic zone with the rest of colon with faeces. Results No tumours appeared in the dimethylhydrazine-free group. The percentage tumoral area was greater in the perianastomotic zone compared to tumours which had developed in the rest of colon with faeces (p = 0.014). Conclusion We found a cocarcinogenic effect due to the creation of an anastomosis, when using an experimental model of colonic carcinogenesis induced by DMH in rats.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Simultaneous irradiation of fibroblasts and carcinoma cells repress the secretion of soluble factors able to stimulate carcinoma cell migration.

Stroma mediated wound healing signals may share similarities with the ones produced by tumor's microenvironment and their modulation may impact tumor response to the various anti-cancer treatments including radiation therapy. Therefore we conducted this study, to assess the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts irrespective of their RhoB status do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms which are TGF-β1 and MMP-mediated respectively. Lastly, we found that simultaneous irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This last result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response.

Improvement of pyrolysis oil quality by scrubbing

Diplomityössä tutkittiin kuuman pyrolyysihöyryn puhdistamista haisevista ja kevyistä haihtuvista yhdisteistä. Työn kirjallisuusosassa selvitettiin pyrolyysiöljyn kannattavuutta uusiutuvana energialähteenä. Lisäksi eri pesurityyppejä tarkasteltiin ja ja vertailtiin. Työn kokeellisessa osassa käytettiin kahta erilaista koelaitteistoa. Tuotteen talteenotossa vertailtiin reaktorilämpötilan ja raaka-aineen kosteuden vaikutusta pyrolyysisaantoihin. Komponenttien talteenotossa tutkittiin epästabiilien ja pistävän hajuisten yhdisteiden poistamista kuumasta pyrolyysihöyrystä. Raaka-aineena käytettiin kuusen metsätäh-dehaketta, joka sisältää runsaasti neulasia ja kaarnaa. Kokeet toteutettiin lämpötila-alueella 460 - 520 °C. Koelaitteistot koostuivat kaasun (N2) syöttöjärjestelmään kytketystä kuumasta ja kyl-mästä puolesta. Tuotteen talteenotossa kuuma pyrolyysihöyry jäähdytettiin ja otettiin talteen. Komponenttien talteenotossa tuote kerättiin suodattimelle ja metyleeniklo-ridiloukkuun. Tuotteiden koostumukset analysoitiin kaasukromatokrafilla. Korkeimmat orgaaniset saannot saatiin 480 °C reaktorilämpötilalla ja 8-9 p-% raaka-ainekosteudella. Pyrolyysiveden määrä putosi raaka-aineen kosteutta nostettaessa. Eri reaktorilämpötiloilla ja raaka-ainekosteuksilla ei ollut vaikutusta hiiltosaantoihin. Kaasusaannot (pääosin CO2, CO ja hiilivedyt) olivat noin 10 p-%. Komponenttien talteenotossa suodatin tukkeutui matalissa (< 250 °C) lämpötiloissa. Suodattimelle jäänyt materiaali oli pääosin neulasista ja kaarnasta peräisin olevia uuteaineita (pääosin hartsi- rasvahappoja) ja sokereita. Korkeimmissa lämpötiloissa (> 250 °C) uuteaineet läpäisivät suodattimen paremmin. 250 ja 300 °C:n lämpötiloissa suuri määrä lyhytketjuisia helposti haihtuvia epästabiileja ja haisevia yhdisteitä (ketoneja, furaani- ja furfuraalijohdannaisia jne.) jäi metyleenikloridi- ja metanoliloukkuihin.

Toll-like receptor 5 deficiency exacerbates cardiac injury and inflammation induced by myocardial ischaemia-reperfusion in the mouse.

Myocardial ischaemia-reperfusion (MIR) triggers a sterile inflammatory response important for myocardial healing, but which may also contribute to adverse ventricular remodelling. Such inflammation is initiated by molecular danger signals released by damaged myocardium, which induce innate immune responses by activating toll-like receptors (TLRs). Detrimental roles have been recently reported for TLR2, TLR3 and TLR4. The role of other TLRs is unknown. We therefore evaluated the role of TLR5, expressed at high level in the heart, in the development of myocardial damage and inflammation acutely triggered by MIR. TLR5-/- and wild-type (WT) mice were exposed to MIR (30 min ischaemia, 2 h reperfusion). We measured infarct size, markers of cardiac oxidative stress, myocardial phosphorylation state of mitogen-activated protein (MAP) kinases and AKT, expression levels of chemokines and cytokines in the heart and plasma, as well as cardiac function by echography and conductance volumetry. TLR5-deficient mice had normal cardiac morphology and function under physiological conditions. After MIR, the absence of TLR5 promoted an increase in infarct size and myocardial oxidative stress. Lack of TLR5 fostered p38 phosphorylation, reduced AKT phosphorylation and markedly increased the expression of inflammatory cytokines, whereas it precipitated acute LV (left ventricle) dysfunction. Therefore, contrary to the detrimental roles of TLR2, TLR3 and TLR4 in the infarcted heart, TLR5 is important to limit myocardial damage, inflammation and functional compromise after MIR.

Kuparinuuttoliuosten adsorptiopuhdistus

Työssä tutkittiin laboratorio-olosuhteissa yhdeksän eri adsorbentin sopivuutta kuparinuuttoliuosten adsorptiopuhdistukseen eli savikäsittelyyn. Tarkoituksena oli poistaa uuttoliuoksesta laimentimen ja reagenssin hapettumis- ja hajoamistuotteita. Adsorbenttien sopivuus uuttoliuosten puhdistukseen määritettiin adsorptio-, kinetiikka- ja faasien selkeytymisaikakokeilla. Tehdyissä kokeissa käytettiin sekä hapetettua synteettistä uuttoliuosta että teollisuudesta saatuja autenttisia kuparinuuttoliuoksia. Uuttoreagenssit olivat hydroksi-oksiimeihin perustuvia. Teollisuudesta saaduista käytetyistä adsorbenteista eluoidun liuoksen kaasugromatografi- ja massaspektrometrianalyysissä ei havaittu selviä merkkejä reagenssin tai laimentimen hajoamistuotteista. Savikäsittely lyhensi faasien selkeytymisaikaa merkittävästi. Bentoniittipohjaisten savien havaittiin soveltuvan parhaiten uuttoliuosten adsorptiopuhdistukseen. IR-analyysin perusteella niiden pinnalle adsorboitui myös eniten karbonyyliryhmiä sisältäviä yhdisteitä. Faasien selkeytymisajan havaittiin huonontuvan itsestään liuosten seistessä savikäsittelyn jälkeen. Noin vuorokauden jälkeen selkeytymisaika tasaantui tasolle, joka oli kuitenkin huomattavasti parempi kuin käsittelemättömällä liuoksella. Savikäsittelyssä käytettävän saven optimimäärä oli noin 1 p-%:a ja sekoitusaika muutamia minuutteja. Savikäsittelyllä ei ollut merkittävää vaikutusta kuparinuuton kinetiikkaan. Työssä tutkittiin laboratorio-olosuhteissa yhdeksän eri adsorbentin sopivuutta kuparinuuttoliuosten adsorptiopuhdistukseen eli savikäsittelyyn. Tarkoituksena oli poistaa uuttoliuoksesta laimentimen ja reagenssin hapettumis- ja hajoamistuotteita. Adsorbe

The role of ubiquitin specific peptidase 15 (USP 15) in human glioblastoma

Glioblastoma (GBM) is the most common and most aggressive malignant primary brain tumour. Despite the aggressiveness of the applied therapy, the prognosis remains poor with a median survival to of about 15 months. It is important to identify new candidate genes that could have clinical application in this disease. Previous gene expression studies from human GBM samples in our laboratory, revealed Ubiquitin Specific Peptidase 15 (USP15) as a gene with low expression, significantly associated with genomic deletions of the chromosomal region encompassing the USP15 locus. USP15 belongs to the ubiquitin-specific protease (USPs) family of which the main role is the reversion of ubiquitination and thereby stabilization of substrates. Previously, USP15 has been suggested to have a tumour suppressor function via its substrates APC and Caspase 3. We established GBM cell lines that stably express USP15 wt or its catalytic mutant. USP15 expression impairs cell growth by inhibiting cell cycle progression. On the other hand USP15 depletion in GBM cell lines induces cell cycle progression and proliferation. In order to identify the molecular pathways in which USP15 is implicated we aimed to identify protein-binding partners in the GBM cell line LN-229 by Mass spectrometry. As a result we identified eight new proteins that interact with USP15. These proteins are involved in important cellular processes like cytokinesis, cell cycle, cellular migration, and apoptosis. Three of these protein interactions were confirmed by co-immunoprecipitation in four GBM cell lines LN-229, LN428, LN18, LN-Z308. One of the binding proteins is HECTD1 E3 ligase of which the murine homologue promotes the APC-Axin interaction to negatively regulate the Wnt pathway. USP15 can de-ubiquitinate HECTD1 in the LN229 cell line while its depletion led to decrease of HECTD1 in GBM cell lines suggesting stabilizing role for USP15. Moreover, HECTD1 stable expression in LN229 inhibits cell cycle, while its depletion induces cell cycle progression. These results suggest that the USP15-HECTD1 interaction might enhance the antiproliferative effect of HECTD1 in GBM cell lines. Using the TOPflash/FOPflash luciferase system we showed that HECTD1 and USP15 overexpression can attenuate WNT pathway activity, and decrease the Axin2 expression. These data indicate that this new protein interaction of USP15 with HECTD1 results in negative regulation of the WNT pathway in GBM cell lines. Further investigation of the regulation of this interaction or of the protein binding network of HECTD1 in GBM may allow the discovery of new therapeutic targets. Finally PTPIP51 and KIF15 are the other two identified protein partners of USP15. These two proteins are involved in cell proliferation and their depletion in LN-229 cell line led to induction of cell cycle progression. USP15 displays a stabilizing role for them. Hence, these results show that the tumour suppressive role of USP15 in GBM cell line via different molecular mechanisms indicating the multidimensional function of USP15. Résumé Le glioblastome (GBM) est la tumeur primaire la plus fréquente et la plus agressive du cervau caractérisée par une survie médiane d'environ à 15 mois. De précédant travaux effectués au sein de notre laboratoire portant sur l'étude de l'expression de gènes pour des échantillons humains de GBM ont montré que le gène Ubiquitin Specific Peptidase 15 (USP1S) était significativement associée à une délétion locales à 25% des cas. Initialement, les substrats protéiques APC et CaspaseS de USP15 ont conduit à considérer cette protéine comme un suppresseur de tumeur. USP15 appartient à la famille protèsse spécifique de l'ubiquitine (USPs) dont le rôle principal est la réversion de l'ubiquitination et la stabilisation de substrats. Par conséquent, nous avons établi des lignées de cellules de glioblastome qui expriment de manière stable USP15 ou bien son mutant catalytique. Ainsi, nous avons ainsi démontré que l'expression de l'USP15 empêche la croissance cellulaire en inhibant la progression du cycle cellulaire. Inversement, la suppression de l'expression du gène USP15 dans les lignées cellulaires de glioblastome induit la progression du cycle cellulaire et la prolifération. Afin d'identifier les voies moléculaires dans lesquelles sont impliquées USP15, nous avons cherché à identifier les partenaires de liaisons protéiques par spectrométrie de masse dans la lignée cellulaire LN-229. Ainsi, huit nouvelles protéines interagissant avec USP15 ont été identifiées dont la ligase E3 HECTD1. L'homologue murin de Hectdl favorise l'interaction APC-Axin en régulant négativement la voie de signalisation de Wnt. USP15 interagit en désubiquitinant HECTD1 dans la lignée cellulaire LN-229 et provoque ainsi l'atténuation de l'activité de cette voie de signalisation. En conclusion, HECTD1, en interagissant avec USP15, joue un rôle de suppresseur de tumeur dans les lignées cellulaire de GBM.

Melanization and pathogenicity in Tenebrio molitor and Pacifastacus leniusculus by Aeromonas hydrophila.

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

BACKGROUND: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. RESULTS: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. CONCLUSIONS: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action.

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

What Role Does Sleep Play in Weight Gain in the First Semester of University?

We hypothesized that shorter sleep durations and greater variability in sleep patterns are associated with weight gain in the first semester of university. Students (N = 132) completed daily sleep diaries for 9 weeks, completed the MEQ (chronotype) and CES-D (depressed mood) at week 9, and self-reported weight/height (weeks 1 & 9). Mean and variability scores were calculated for sleep duration (TST, TSTv), bedtime (BT, BTv), and wake time (WT, WTv). An initial hierarchical regression evaluated (block 1) sex, ethnicity; (block 2) depressed mood, chronotype; (block 3) TST; (block 4) BT, WT; and (block 5; R(2) change = 0.09, p = 0.005) TSTv, BTv, WTv with weight change. A sex-by-TSTv interaction was found. A final model showed that ethnicity, TST, TSTv, and BTv accounted for 31% of the variance in weight change for males; TSTv was the most significant contributor (R(2) change = 0.21, p < 0.001). Daily variability in sleep duration contributes to males' weight gain. Further investigation needs to examine sex-specific outcomes for sleep and weight.