An activation-independent role of transcription factors in insulator function.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Effect on renal function of tenofovir co-administered with either efavirenz or boosted lopinavir or boosted atazanavir: the Swiss HIV Cohort Study

Reduced re'nal function has been reported with tenofovir disoproxil fumarate (TDF). It is not clear whether TDF co-administered with a boosted protease inhibitor (PI) leads to a greater decline in renal function than TDF co-administered with a non-nucleoside reverse transcriptase inhibitor (NNRTI).Methods: We selected ail antiretroviral therapy-naive patients in the Swiss HIV Cohort Study (SHCS) with calibrated or corrected serum creatinine measurements starting antiretroviral therapy with TDF and either efavirenz (EFV) or the ritonavir-boosted PIs, lopinavir (LPV/r) or atazanavir (ATV/r). As a measure of renal function, we used the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation to estimate the glomerular filtration rate (eGFR). We calculated the difference in eGFR over time between two therapies using a marginal model for repeated measures. In weighted analyses, observations were weighted by the product of their point of treatment and censoring weights to adjust for differences both in the sort of patients starting each therapy and in the sort of patients remaining on each therapy over time.Results: By March 2011, 940 patients with at least one creatinine measurement on a first therapy with either TDF and EFV (n=484), TDF and LPVlr (n=269) or TDF and ATV/r (n=187) had been followed for a median of 1. 7, 1.2 and 1.3 years, respectively. Table 1 shows the difference in average estimated GFR (eGFR) over time since starting cART for two marginal models. The first model was not adjusted for potential confounders; the second mode! used weights to adjust for confounders. The results suggest a greater decline in renal function during the first 6 months if TDF is used with a PI rather than with an NNRTI, but no further difference between these therapies after the first 6 months. TDF and ATV/r may lead to a greater decline in the first 6 months than TDF and LPVlr.Conclusions: TDF co-administered with a boosted PI leads to a greater de cline in renal function over the first 6 months of therapy than TDF co-administered with an NNRTI; this decline may be worse with ATV/r than with LPV/r.

Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon gamma (IFNgamma) production, suggesting sustained signaling. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DNAX-activating protein of 10 kDa (DAP-10) and killer cell activating receptor-associated protein/DNAX-activating protein of 12 kDa (KARAP/DAP-12). That is likely the consequence of constitutive NKG2D engagement and signaling, since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Thus, the chronic exposure to tumor cells expressing NKG2D ligand alters NKG2D signaling and may facilitate the evasion of tumor cells from NK cell reactions.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

Connexin-36 contributes to control function of insulin-producing cells.

Connexin-36 (Cx36) is a gap junction protein expressed by the insulin-producing beta-cells. We investigated the contribution of this protein in normal beta-cell function by using a viral gene transfer approach to alter Cx36 content in the insulin-producing line of INS-1E cells and rat pancreatic islets. Transcripts for Cx43, Cx45, and Cx36 were detected by reverse transcriptase-PCR in freshly isolated pancreatic islets, whereas only a transcript for Cx36 was detected in INS-1E cells. After infection with a sense viral vector, which induced de novo Cx36 expression in the Cx-defective HeLa cells we used to control the transgene expression, Western blot, immunofluorescence, and freeze-fracture analysis showed a large increase of Cx36 within INS-1E cell membranes. In contrast, after infection with an antisense vector, Cx36 content was decreased by 80%. Glucose-induced insulin release and insulin content were decreased, whether infected INS-1E cells expressed Cx36 levels that were largely higher or lower than those observed in wild-type control cells. In both cases, basal insulin secretion was unaffected. Comparable observations on basal secretion and insulin content were made in freshly isolated rat pancreatic islets. The data indicate that large changes in Cx36 alter insulin content and, at least in INS-1E cells, also affect glucose-induced insulin release.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Immune regulation and function of the NOD-like receptors NLRC5 and NLRP3

AbstractThe vertebrate immune system is composed of the innate and the adaptive branches. Innate immune cells represent the first line of defense and detect pathogens through pattern recognition receptors (PRRs), detecting evolutionary conserved pathogen- and danger- associated molecular patterns. Engagement of these receptors initiates the inflammatory response, but also instructs antigen-specific adaptive immune cells. NOD-like receptors (NLRs) are an important group of PRRs, leading to the production of inflammatory mediators and favoring antigen presentation to Τ lymphocytes through the regulation of major histocompatibility complex (MHC) molecules.In this work we focused our attention on selected NOD-like receptors (NLRs) and their role at the interface between innate and adaptive immunity. First, we describe a new regulatory mechanism controlling IL-1 production. Our results indicate that type I interferons (IFNs) block NLRP1 and NLRP3 inflammasome activity and interfere with LPS-driven proIL-Ια and -β induction. As type I IFNs are produced upon viral infections, these anti-inflammatory effects of type I IFN could be relevant in the context of superinfections, but could also help explaining the efficacy of IFN-β in multiple sclerosis treatment.The second project addresses the role of a novel NLR family member, called NLRC5. The function of this NLR is still matter of debate, as it has been proposed as both an inhibitor and an activator of different inflammatory pathways. We found that the expression of this protein is restricted to immune cells and is positively regulated by IFNs. We generated Nlrc5-deficient mice and found that this NLR plays an essential role in Τ, NKT and, NK lymphocytes, in which it drives the expression of MHC class I molecules. Accordingly, we could show that CD8+ Τ cell-mediated killing of target lymphocytes lacking NLRC5 is strongly impaired. Moreover, NLRC5 expression was found to be low in many lymphoid- derived tumor cell lines, a mechanism that could be exploited by tumors to escape immunosurveillance.Finally, we found NLRC5 to be involved in the production of IL-10 by CD4+ Τ cells, as Nlrc5- deficient Τ lymphocytes produced less of this cytokine upon TCR triggering. In line with these observations, Mrc5-deficient CD4+ Τ cells expanded more than control cells when transferred into lymphopenic hosts and led to a more rapid appearance of colitis symptoms. Therefore, our work gives novel insights on the function of NLRC5 by using knockout mice, and strongly supports the idea that NLRs direct not only innate, but also adaptive immune responses.

IV thrombolysis and renal function.

OBJECTIVE: To investigate the association of renal impairment on functional outcome and complications in stroke patients treated with IV thrombolysis (IVT). METHODS: In this observational study, we compared the estimated glomerular filtration rate (GFR) with poor 3-month outcome (modified Rankin Scale scores 3-6), death, and symptomatic intracranial hemorrhage (sICH) based on the criteria of the European Cooperative Acute Stroke Study II trial. Unadjusted and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Patients without IVT treatment served as a comparison group. RESULTS: Among 4,780 IVT-treated patients, 1,217 (25.5%) had a low GFR (<60 mL/min/1.73 m(2)). A GFR decrease by 10 mL/min/1.73 m(2) increased the risk of poor outcome (OR [95% CI]): (ORunadjusted 1.20 [1.17-1.24]; ORadjusted 1.05 [1.01-1.09]), death (ORunadjusted 1.33 [1.28-1.38]; ORadjusted 1.18 [1.11-1.249]), and sICH (ORunadjusted 1.15 [1.01-1.22]; ORadjusted 1.11 [1.04-1.20]). Low GFR was independently associated with poor 3-month outcome (ORadjusted 1.32 [1.10-1.58]), death (ORadjusted 1.73 [1.39-2.14]), and sICH (ORadjusted 1.64 [1.21-2.23]) compared with normal GFR (60-120 mL/min/1.73 m(2)). Low GFR (ORadjusted 1.64 [1.21-2.23]) and stroke severity (ORadjusted 1.05 [1.03-1.07]) independently determined sICH. Compared with patients who did not receive IVT, treatment with IVT in patients with low GFR was associated with poor outcome (ORadjusted 1.79 [1.41-2.25]), and with favorable outcome in those with normal GFR (ORadjusted 0.77 [0.63-0.94]). CONCLUSION: Renal function significantly modified outcome and complication rates in IVT-treated stroke patients. Lower GFR might be a better risk indicator for sICH than age. A decrease of GFR by 10 mL/min/1.73 m(2) seems to have a similar impact on the risk of death or sICH as a 1-point-higher NIH Stroke Scale score measuring stroke severity.

Long-term fluctuation of relative afferent pupillary defect in subjects with normal visual function.

PURPOSE: To determine whether the relative afferent pupillary defect (RAPD) remains constant over time in normal subjects. METHODS: Seventeen normal subjects were tested with infrared pupillography and automated perimetry in four sessions over 3 years. The changes in RAPD and visual field asymmetry between testing sessions were compared. RESULTS: The range of RAPD was 0.0 to 0.3 log unit, and the difference in the mean deviation between the eyes on automated static perimetry was 0 to 3 dB. Eight subjects repeatedly had an RAPD in the same eye. There was no correlation between the RAPD and the visual field asymmetry at the same visit. Changes in the magnitude of the RAPD between any two sessions were typically small (median, 0.08 log unit; 25th percentile, 0.04 log unit; 75th percentile, 0.15 log unit). CONCLUSIONS: Some normal subjects may show a persistent but small RAPD in the absence of detectable pathologic disease. Therefore, an isolated RAPD in the range of 0.3 log unit that is not associated with any other significant historical or clinical finding should probably be considered benign.

Regulation of blood pressure and renal function by NCC and ENaC: lessons from genetically engineered mice.

The activity of the thiazide-sensitive Na(+)/Cl(-) cotransporter (NCC) and of the amiloride-sensitive epithelial Na(+) channel (ENaC) is pivotal for blood pressure regulation. NCC is responsible for Na(+) reabsorption in the distal convoluted tubule (DCT) of the nephron, while ENaC reabsorbs the filtered Na(+) in the late DCT and in the cortical collecting ducts (CCD) providing the final renal adjustment to Na(+) balance. Here, we aim to highlight the recent advances made using transgenic mouse models towards the understanding of the regulation of NCC and ENaC function relevant to the control of sodium balance and blood pressure. We thus like to pave the way for common mechanisms regulating these two sodium-transporting proteins and their potential implication in structural remodeling of the nephron segments and Na(+) and Cl(-) reabsorption.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems.

To sense myriad environmental odors, animals have evolved multiple, large families of divergent olfactory receptors. How and why distinct receptor repertoires and their associated circuits are functionally and anatomically integrated is essentially unknown. We have addressed these questions through comprehensive comparative analysis of the Drosophila olfactory subsystems that express the ionotropic receptors (IRs) and odorant receptors (ORs). We identify ligands for most IR neuron classes, revealing their specificity for select amines and acids, which complements the broader tuning of ORs for esters and alcohols. IR and OR sensory neurons exhibit glomerular convergence in segregated, although interconnected, zones of the primary olfactory center, but these circuits are extensively interdigitated in higher brain regions. Consistently, behavioral responses to odors arise from an interplay between IR- and OR-dependent pathways. We integrate knowledge on the different phylogenetic and developmental properties of these receptors and circuits to propose models for the functional contributions and evolution of these distinct olfactory subsystems.