Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Distinct subcellular localization of beta-tubulin epitopes in the adult mouse brain.

A panel of monoclonal antibodies specific of alpha-tubulin (TU-01, TU-09) and beta-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU-13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of beta-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of beta-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of beta-tubulin by interacting protein(s) in dendrites and axons.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Peroxisome proliferator-activated receptors: insight into multiple cellular functions.

Peroxisome proliferator-activated receptors, PPARs, (NR1C) are nuclear hormone receptors implicated in energy homeostasis. Upon activation, these ligand-inducible transcription factors stimulate gene expression by binding to the promoter of target genes. The different structural domains of PPARs are presented in terms of activation mechanisms, namely ligand binding, phosphorylation, and cofactor interaction. The specificity of ligands, such as fatty acids, eicosanoids, fibrates and thiazolidinediones (TZD), is described for each of the three PPAR isotypes, alpha (NR1C1), beta (NR1C2) and gamma (NR1C3), so as the differential tissue distribution of these isotypes. Finally, general and specific functions of the PPAR isotypes are discussed, namely their implication in the control of inflammatory responses, cell proliferation and differentiation, the roles of PPARalpha in fatty acid catabolism and of PPARgamma in adipogenesis.

Penicillin tolerance among Beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11,7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbo-hydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

Eurytrema coelomaticum: influence of the infection on the reproduction and nucleic acids contents in the albumen gland and ovotestis of Bradybaena similaris

The number of eggs laid per snail in Bradybaena similaris and the nucleic acids (DNA and RNA) in the albumen gland and ovotestis were quantified in snails infected with sporocysts of the digenetic trematode Eurytrema coelomaticum. The total number of eggs laid per mollusc was reduced by 96.32% at the end of the larval development. The DNA concentration increased by 700% and the RNA concentration was reduced by 8,38% by the time when the daughter sporocysts of E. coelomaticum were released from B. similaris. The relation between these values and the inhibition of the reproduction observed in infected molluscs is discussed.

Teichuronic acid operon of Bacillus subtilis 168.

Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP-glucuronate. Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls. The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription. Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer. Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.

The influence of sugars and amino acids on the blood-feeding behaviour, oviposition and longevity of laboratory colony of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae, Phlebotominae)

Schneider's Drosophila medium, a complex amino acid rich medium was tested alone and with seven different sugars for some aspects of the biology of Lutzomyia longipalpis. Statistically significant results were obtained when sucrose was used alone, indicating that among the sugars tested, this is still the most suitable and practical one for the maintenance of L. longipalpis colonies. However, the addition of Schneider's medium to a pool of different sugars, was suggested to be related with the acceptance of the first and second blood meals and to longevity, these being, obviously, quite relevant aspects when tansmission experiments are contemplated.

Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.

Biased V beta usage in immature thymocytes is independent of DJ beta proximity and pT alpha pairing.

During thymus development, the TCR beta locus rearranges before the TCR alpha locus. Pairing of productively rearranged TCR beta-chains with an invariant pT alpha chain leads to the formation of a pre-TCR and subsequent expansion of immature pre-T cells. Essentially nothing is known about the TCR V beta repertoire in pre-T cells before or after the expression of a pre-TCR. Using intracellular staining, we show here that the TCR V beta repertoire is significantly biased at the earliest developmental stage in which VDJ beta rearrangement has occurred. Moreover (and in contrast to the V(H) repertoire in immature B cells), V beta repertoire biases in immature T cells do not reflect proximity of V beta gene segments to the DJ beta cluster, nor do they depend upon preferential V beta pairing with the pT alpha chain. We conclude that V gene repertoires in developing T and B cells are controlled by partially distinct mechanisms.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Regulated expression of GLUT2 in diabetes studied in transplanted pancreatic beta cells.

GLUT2 disappearance is a marker of the beta cell glucose-unresponsiveness associated with diabetes. Understanding the factor(s) leading to this dysfunction may shed light on pathogenesis of diabetes. Since the regulation of GLUT2 expression in diabetes can so far only be studied in in vivo experiments, we developed a novel experimental approach to study the genetic regulation of GLUT2 in diabetes. By encapsulating islets or cell lines in semi-permeable membranes, these cells can be exposed to the diabetic environment of rats or mice and can be retrieved for analysis of GLUT2 expression and for the change in the secretory response to glucose. Immunocytochemical analysis of transporter expression reveals changes in protein expression while transcriptional analysis of GLUT2 gene expression could be performed in cells transfected with promoter-reporter gene constructs. Using this last approach we hope to be able to characterize the promoter regions involved in the beta cell- and diabetes-specific regulation of GLUT2 expression and possibly to determine which factors are responsible for this regulation.

Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.

Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA.

Allogeneic beta-islet cells correct diabetes and resist immune rejection.

Allogeneic MHC-incompatible organ or cell grafts are usually promptly rejected by immunocompetent hosts. Here we tested allogeneic beta-islet cell graft acceptance by immune or naive C57BL/6 mice rendered diabetic with streptozotocin (STZ). Fully MHC-mismatched insulin-producing growth-regulated beta-islet cells were transplanted under the kidney capsule or s.c. Although previously or simultaneously primed mice rejected grafts, STZ-treated diabetic mice accepted islet cell grafts, and hyperglycemia was corrected within 2-4 weeks in absence of conventional immunosuppression. Allogeneic grafts that controlled hyperglycemia expressed MHC antigens, were not rejected for >100 days, and resisted a challenge by allogeneic skin grafts or multiple injections of allogeneic cells. Importantly, the skin grafts were rejected in a primary fashion by the grafted and corrected host, indicating neither tolerization nor priming. Such strictly extralymphatic cell grafts that are immunologically largely ignored should be applicable clinically.

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom