Loss of hippocampal neuronal nitric oxide synthase contributes to the stress-related deficit in learning and memory

Nitric oxide (NO) has been involved in many pathophysiological brain processes. However, the exact role of NO in the cognitive deficit associated to chronic stress exposure has not been elucidated. In this study, we investigated the participation of hippocampal NO production and their regulation by protein kinase C (PKC) in the memory impairment induced in mice subjected to chronic mild stress model (CMS). CMS mice showed a poor learning performance in both open field and passive avoidance inhibitory task respect to control mice. Histological studies showed a morphological alteration in the hippocampus of CMS mice. On the other hand, chronic stress induced a diminished NO production by neuronal nitric oxide synthase (nNOS) correlated with an increment in gamma and zeta PKC isoenzymes. Partial restoration of nNOS activity was obtained after PKC activity blockade. NO production by inducible nitric oxide synthase isoform was not detected. The magnitude of oxidative stress, evaluated by reactive oxygen species production, after excitotoxic levels of NMDA was increased in hippocampus of CMS mice. Moreover, ROS formation was higher in the presence of nNOS inhibitor in both control and CMS mice. Finally, treatment of mice with nNOS inhibitors results in behavioural alterations similar to those observed in CMS animals. These findings suggest a novel role for nNOS showing protective activity against insults that trigger tissue toxicity leading to memory impairments.     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.     

Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.     

Antagonistic Roles of PP2A-Pab1 and Etd1 in the Control of Cytokinesis in Fission Yeast

In Schizosaccharomyces pombe, Etd1 is a positive regulator of the septation initiation network (SIN), a conserved GTPase-regulated kinase cascade that triggers cytokinesis. Here we show that a mutation in the pab1 gene, which encodes the B-regulatory subunit of the protein phosphatase 2A (PP2A), suppresses mutations in the etd1 gene. Etd1 is required for the function of the GTPase Spg1, a key regulator of SIN signaling. Interestingly, the loss of Pab1 function restored the activity of Spg1 in Etd1-deficient cells. This result suggests that PP2A-Pab1–mediated dephosphorylation inhibits Spg1, thus antagonizing Etd1 function. The loss of pab1 function also rescues the lethality of mutants of other genes in the SIN cascade such as mob1, sid1, and cdc11. Two-hybrid assays indicate that Pab1 physically interacts with Mob1, Sid1, Sid2, and Cdc11, suggesting that the phosphatase 2A B-subunit is a component of the SIN complex. Together, our results indicate that PP2A-Pab1 plays a novel role in cytokinesis, regulating SIN activity at different levels. Pab1 is also required to activate polarized cell growth. Thus, PP2A-Pab1 may be involved in coordinating polar growth and cytokinesis.THE fission yeast Schizosaccharomyces pombe is a leading experimental model for eukaryotic cytokinesis (Bathe and Chang 2009; Pollard and Wu 2010). Fission yeast cells grow in a polarized manner by elongation at the cell ends and divide during cytokinesis by the action of a contractile actomyosin ring assembled in the middle of the cell (Snell and Nurse 1993). At the end of mitosis, when nuclear separation has been completed, actomyosin ring constriction is triggered by the septation initiation network (SIN). This signal transduction cascade is composed of the GTPase Spg1 and three protein kinases—Cdc7, GC-kinase Sid1, and NDR-kinase Sid2 in their presumed order of action—and the associated proteins Cdc14 with Sid1 and Mob1 with Sid2. These proteins are all located at the spindle pole body (SPB) during mitosis on a scaffold composed of the coiled-coil proteins Sid4 and Cdc11 (Krapp et al. 2004). The Sid2-Mob1 protein kinase complex is thought to transmit the division signal from the SPB to the actomyosin ring since it also associates at the division site during septation (Krapp and Simanis 2008). The SIN triggers actomyosin ring contraction coordinated with the synthesis of the primary and secondary septa that will form the new cell wall (Krapp et al. 2004; Wolfe and Gould 2005). The small GTPase Rho1 is known to promote cell-wall formation at the division site by stimulation of Cps1p/Drc1 1,3-β-glucan synthase (Le Goff et al. 1999), but the mechanism remains unclear.SIN activity is tightly regulated during the cell cycle to ensure proper coordination of mitosis and cytokinesis. Mutants that negatively affect SIN function undergo nuclear division in the absence of septation, while increased SIN activity induces septation in interphase cells (Krapp and Simanis 2008). Regulation of the SIN is complex, involving multiple, partially redundant mechanisms, but the nucleotide status of the Ras superfamily small GTPase, Spg1, represents a key step in SIN activity (Lattmann et al. 2009). Cdc16 and Byr4 form a two-component GTPase-activating protein (GAP) for Spg1 that inhibits its activity (Furge et al. 1998; Cerutti and Simanis 1999). Proteins acting as a guanine nucleotide-exchange factor (GEF) for this GTPase have not been identified. In the budding yeast Saccharomyces cerevisiae, the pathway analogous to the SIN is known as the mitotic exit network (MEN) (reviewed in Krapp and Simanis 2008). Contact between the SPB-localized GTPase Tem1 (the Spg1 homolog) with its putative GEF Lte1, which is present only within the bud, has been proposed as a mechanism to ensure that mitotic exit occurs only after the spindle has oriented correctly (Bardin et al. 2000; Pereira et al. 2000). Bfa1-Bub2 (the Cdc16-Byr4 equivalent) are negative regulators of the MEN, acting as a two-component GAP for Tem1 (Geymonat et al. 2002).Etd1 was identified in a genetic screen searching for new regulators of the S. pombe cell division cycle (Jimenez and Oballe 1994). Further characterization indicated that Etd1 acts as a positive regulator of the SIN (Daga et al. 2005). A recent study has established a key role for Etd1 in the timing of cytokinesis via the regulation of Spg1, acting as a potential homolog of budding yeast Lte1 (Garcia-Cortes and McCollum 2009). Loss of Etd1 function can be suppressed by mutations in a number of genes, some of which are involved in morphogenesis (Jimenez and Oballe 1994). Here we show that one of the mutations that bypass the requirement for etd1 in cytokinesis affects the activity of pab1, which encodes the protein phosphatase 2A (PP2A) regulatory subunit B. The characterization of Pab1 and pab1 mutants described in this study reveals a novel role for PP2A-Pab1 in SIN regulation and provides new insight into the mechanism by which Etd1 might regulate SIN signaling. We also show that Pab1 participates in activation of the morphological pathway, suggesting a role for PP2A-Pab1 in the coordination of cytokinesis and morphogenesis.     

Role of TLR9 in anti-nucleosome and anti-DNA antibody production in lpr mutation-induced murine lupus

Systemic lupus erythematosus is characterized by the production of autoantibodies directed against nuclear Ags, including nucleosome and DNA. TLR9 is thought to play a role in the production of these autoantibodies through the capacity of nuclear immunogenic particles to interact both with BCR and TLR9. To determine the role of TLR9 in SLE, C57BL/6-lpr/lpr-TLR9(-/-) and TLR9(+/+) mice were analyzed. The abrogation of TLR9 totally impaired the production of anti-nucleosome Abs, whereas no difference was observed in the frequency of anti-dsDNA autoantibodies whose titer was strikingly higher in TLR9(-/-) mice. In addition a higher rate of mesangial proliferation was observed in the kidney of TLR9-deficient animals. These results indicate that in C57BL/6-lpr/lpr mice, TLR9 is absolutely required for the anti-nucleosome Ab response but not for anti-dsDNA Ab production which is involved in mesangial proliferation.     

Early evidence of lymphoproliferative disorder: post-transplant monitoring of Epstein-Barr infection in adult and pediatric patients

Transplant patients are at high risk of post-transplant lymphoproliferative disorder (PTLD). A strong correlation between Epstein-Barr virus (EBV) and PTLD is observed in pediatric patients with primary infection after transplant. Because many patients have responded to reversal of immunosuppressive therapy, an early identification of EBV is essential for the reduction of immunosuppression and/or introduction of antiviral therapy to prevent PTLD. Polymerase chain reaction (PCR) is a specific and sensitive method to identify EBV DNA in blood. The aim of our study was to establish a protocol for monitoring EBV infection in transplanted patients for early identification those at high risk of PTLD. Viral presence in peripheral blood leukocytes (PBL) and serum samples was revealed by Nested PCR; positive specimens were quantified with Real Time PCR (RT-PCR). DNA in PBL was observed in 12 cases and 6 showed EBV in sera. Quantitative analysis showed a wide range of EBV DNA copies in leukocytes that were higher than in sera. Two patients displayed high viral load values in both PBL and sera associated with clinical evidence of PTLD. Our data suggest that the study of the EBV load represents an essential approach in the diagnosis of PTLD and the analysis of serum samples could provide useful information in the post-transplant monitoring of high-risk patients.     

Anti-Mullerian hormone plasma concentration in prepubertal ewe lambs as a predictor of their fertility at a young age

ABSTRACT: BACKGROUND: In mammals, the ovarian follicular reserve is highly variable between individuals and impacts strongly on ovarian function and fertility. Nowadays, the best endocrine marker of this reserve in human, mouse and cattle is the anti-Mullerian hormone (AMH). The objectives of this work were to determine whether AMH could be detected in the plasma of prepubertal ewe lambs and to assess its relationship with their fertility at a young age. RESULTS: Plasma was taken from 76 Rasa Aragonesa ewe lambs at 3.6 months of age for AMH determination. Simultaneously, 600 IU equine chorionic gonadotropin (eCG) was administered and the number of ovulations recorded 6 days later. AMH was detected in 93 % of the lambs, and the concentrations were about 3-4-fold higher in ovulating than in non-ovulating lambs (P < 0.004). Ewes aged around 10 months were mated, giving an overall fertility of 29 %, and those failing to conceive were mated again 4 months later. Fertility at first mating was significantly correlated with plasma AMH concentration at 3.6 months (Spearman's rho = 0.34; P < 0.01). To use plasma AMH concentration as a screening test, a value of 97 pg/mL was determined as the optimum cutoff value to predict fertility at first mating (sensitivity = 68.2 %; specificity = 72.2 %). Fertility at first mating was 34.8 percentage points higher in ewe lambs with an AMH [greater than or equal to] 97 pg/mL than in those with lower AMH concentrations (50 % vs. 15 %; P < 0.001). CONCLUSIONS: Plasma AMH concentration might be a reliable marker of the ovarian status of prepubertal ewe lambs, reflecting their ability to respond to eCG stimulation. A single AMH measurement performed on ewe lambs early in age could be useful to select for replacement ewes with a higher predicted fertility at first mating.     

Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport

Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.