A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

17beta-Estradiol enhances neuronal differentiation of mouse embryonic stem cells

Existing protocols show a variety in the percentage of neurons that can be generated from mouse embryonic stem (ES) cells. In the current study, we compared effects of various differentiating conditions, including gelatin and poly-l-ornithine/fibronectin coatings, and NGF and 17beta-estradiol treatments on the total yield of neurons, as well as, neurite growth and branching. Here, we show that combination of fibronectin coating with 17beta-estradiol increased number of generated neurons over 50%. Poly-l-ornithine/fibronectin increased the percent of neurons in all cultures, suggesting its direct influence on neurogenesis. Addition of 17beta-estradiol reduced mean neurite length in culture, but significantly increased branching. Our results indicate a substrate-dependent regulation of estrogen-induced ES cells differentiation into neuronal cells.     

Structural analysis of the human Golgi-associated plant pathogenesis related protein GAPR-1 implicates dimerization as a regulatory mechanism

The plant pathogenesis related proteins group 1 (PR-1) and a variety of related mammalian proteins constitute a PR-1 protein family that share sequence and structural similarities. GAPR-1 is a unique family member as thus far it is the only PR-1 family member that is not co-translationally targeted to the lumen of the endoplasmic reticulum before trafficking to either vacuoles or secretion. Here we report that GAPR-1 may form dimers in vitro and in vivo, as determined by yeast two-hybrid screening, biochemical and biophysical assays. The 1.55A crystal structure demonstrates that GAPR-1 is structurally homologous to the other PR-1 family members previously solved (p14a and Ves V 5). Through an examination of inter-molecular interactions between GAPR-1 molecules in the crystal lattice, we propose a number of the highly conserved amino acid residues of the PR-1 family to be involved in the regulation of dimer formation of GAPR-1 with potential implications for other PR-1 family members. We show that mutagenesis of these conserved amino acid residues leads to a greatly increased dimer population. A recent report suggests that PR-1 family members may exhibit serine protease activity and further examination of the dimer interface of GAPR-1 indicates that a catalytic triad similar to that of serine proteases may be formed across the dimer interface by residues from both molecules within the dimer.     

Consequences of cytotoxic T-lymphocyte escape: common escape mutations in simian immunodeficiency virus are poorly recognized in naive hosts

Cytotoxic T lymphocytes (CTL) are associated with control of immunodeficiency virus infection but also select for variants that escape immune recognition. Declining frequencies of epitope-specific CTL frequencies have been correlated with viral escape in individual hosts. However, escape mutations may give rise to new epitopes that could be recognized by CTL expressing appropriate T-cell receptors and thus still be immunogenic when escape variants are passed to individuals expressing the appropriate major histocompatibility complex class I molecules. To determine whether peptide ligands that have been altered through escape can be immunogenic in new hosts, we challenged naïve, immunocompetent macaques with a molecularly cloned simian immunodeficiency virus (SIV) bearing common escape mutations in three immunodominant CTL epitopes. Responses to the altered peptides were barely detectable in fresh samples at any time after infection. Surprisingly, CTL specific for two of three escaped epitopes could be expanded by in vitro stimulation with synthetic peptides. Our results suggest that some escape variant epitopes evolving in infected individuals do not efficiently stimulate new populations of CTL, either in that individual or upon passage to new hosts. Nevertheless, escape variation may not completely abolish an epitope''s immunogenicity. Moreover, since the mutant epitope sequences did not revert to wild type during the study period, it is possible that low-frequency CTL exerted enough selective pressure to preserve epitope mutations in viruses replicating in vivo.In recent years, there has been increasing interest in AIDS vaccine approaches that elicit cytotoxic T lymphocytes (CTL), which recognize and eliminate cells infected with human immunodeficiency virus (HIV) (26). Unlike antibodies, effective CTL responses can be directed against epitopes derived from any viral protein, raising the possibility that CTL can be targeted to regions that are more conserved than the viral envelope. Current vaccine modalities can elicit potent CTL responses against multiple viral epitopes (25). Indeed, many lines of evidence indicate that cell-mediated immunity plays a major role in control of virus replication. Several studies have suggested an association between certain major histocompatibility complex (MHC) class I and class II alleles and control of viral replication or susceptibility to disease (6, 7, 11, 12, 15-17, 28, 36, 38, 39). CTL are also implicated in the initial control of immunodeficiency virus infection, since they appear in close temporal association with the reduction in peak viremia in both HIV-infected humans (5, 22) and simian immunodeficiency virus (SIV)-infected macaques (23). Antibody-mediated depletion of CD8⁺ cells in infected macaques resulted in dramatically increased virus loads in both acute and chronic infection (14, 27, 37).However, the plasticity of the viral genome also allows the generation of mutants that escape CTL recognition. Certain high-frequency CTL exert intense selective pressure on virus sequences, as revealed by the nearly total extinction of CTL-susceptible sequences from the actively replicating virus population within a few weeks of infection (2, 32). Escape from CTL has been observed in several studies of infected humans (12, 18, 21, 34, 35, 41) and macaques (2, 8, 30, 32, 40). Moreover, one report has shown that an HIV-1 escape mutant can be transmitted vertically (11), while other studies in vaccinated macaques have suggested that the evolution of escape mutants may be associated with a loss of containment of viral replication (4, 31). It therefore seems likely that escape from CTL responses occurs in most infected individuals (32).The apparent ubiquity of CTL escape may greatly complicate the design of CTL-based vaccines. The evolution of escape variants during infection of a single host may play a key part in viral persistence and therefore in the ultimate failure of immune containment and progression to AIDS. However, some investigators have suggested that T-cell receptor repertoires can recognize multiple epitope variants, so that CTL responses can coevolve along with viral escape variants in infected individuals (13). If T-cell receptor populations can recognize new variant epitopes arising within a single host, it seems plausible that variant epitope sequences could also be recognized efficiently in new hosts. Escape could also create “neo-epitopes,” novel sequences that are immunogenic to naïve T cells in individuals expressing the appropriate MHC class I molecules.The most rigorous test of the immunogenicity of epitopes altered through escape is to challenge a fully intact immune system with an escape mutant virus. Therefore, we identified common escape mutations that accumulated in immunodominant epitopes of SIVmac239 in infected macaques expressing the high-frequency MHC class I molecules Mamu-A*01 and Mamu-B*17. Together, these molecules bind three immunodominant CTL epitopes in SIVmac239: Gag_181-189CM9 (CTPYDINQM, Gag CM9) and Tat_28-35SL8 (STPESANL, Tat SL8) are bound by Mamu-A*01, and Nef_165-173IW9 (IRYPKTFGW, Nef IW9) is bound by Mamu-B*17. We have previously shown that the acute-phase response in Mamu-A*01 Mamu-B*17 double-positive macaques is dominated by CTL that recognize these three epitopes (33). We introduced common escape mutations into the SIVmac239 molecular clone and challenged macaques expressing both Mamu-A*01 and Mamu-B*17 with the mutant virus. CTL responses directed against the mutant epitopes were extremely low frequency or undetectable in fresh samples from each of the infected animals. In the absence of these responses, a completely new immunodominance hierarchy was established. Our results suggest it is unlikely that “escaped” epitopes will be recognized in newly infected individuals expressing appropriate MHC class I molecules.     

A cluster of Arabidopsis genes with a coordinate response to an environmental stimulus

Vernalization, the promotion of flowering after prolonged exposure to low temperatures, is an adaptive response of plants ensuring that flowering occurs at a propitious time in the annual seasonal cycle. In Arabidopsis, FLOWERING LOCUS C (FLC), which encodes a repressor of flowering, is a key gene in the vernalization response; plants with high-FLC expression respond to vernalization by downregulating FLC and thereby flowering at an earlier time. Vernalization has the hallmarks of an epigenetically regulated process. The downregulation of FLC by low temperatures is maintained throughout vegetative development but is reset at each generation. During our study of vernalization, we have found that a small gene cluster, including FLC and its two flanking genes, is coordinately regulated in response to genetic modifiers, to the environmental stimulus of vernalization, and in plants with low levels of DNA methylation. Genes encoded on foreign DNA inserted into the cluster also acquire the low-temperature response. At other chromosomal locations, FLC maintains its response to vernalization and imposes a parallel response on a flanking gene. This suggests that FLC contains sequences that confer changes in gene expression extending beyond FLC itself, perhaps through chromatin modification.     

Spatial and Temporal Variation of Enterobacter Genotypes in Sediments and the Underlying Hyporheic Zone of an Agricultural Stream

Population studies of enteric bacteria in an agriculturally impacted stream (Ledbetter Creek, Murray, Kentucky, USA) were conducted over a period of 2 years. Total number of bacteria, cultivated heterotrophic aerobic bacteria, and enteric bacteria showed significant differences between winter and summer. The cultivated numbers of heterotrophic aerobic bacteria and enteric bacteria were significantly more abundant in summer than in winter. The abundance of enteric bacteria was 12.9% in an upwelling zone and 9.8% in a downwelling zone in summer. Most of the enteric bacterial strains isolated on MacConkey agar were assigned to Enterobacter cloacae and E. agglomerans by API 20E and an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (ARDRA) with the enzyme Hpa II. E. cloacae and E. agglomerans genotypes isolated from three hyporheic and gravel bar depth intervals (0-10 cm, 15-25 cm, and 30-40 cm) in summer and fall showed significant spatial variation and were heterogeneously distributed along the stream. Temperature, inorganic nutrients, and occurrence of anoxic zones affected the distribution of enteric bacteria. These techniques can be used as a model to monitor shifts among different species in the stream ecosystem.     

Maternal gestational androgens are associated with decreased juvenile play in white-faced marmosets (Callithrix geoffroyi)

Exposure to androgens during prenatal development shapes both physiological and behavioral developmental trajectories. Notably, in rhesus macaques, prenatal androgen exposure has been shown to increase rough-and-tumble play, a prominent behavioral feature in males during the juvenile period in primates. While macaques are an Old World, polygamous species with marked sexually dimorphic behavior, New World callitrichine primates (marmosets and tamarins) live in cooperative breeding groups and are considered to be socially monogamous and exhibit minimal sexual dimorphism in social play, which suggests that androgen may affect this species in different ways compared to macaques. In addition, we previously described considerable variation in maternal androgen production during gestation in marmosets. Here we tested the association between this variation and variation in offspring rough-and-tumble play patterns in both males and females. We measured testosterone and androstenedione levels in urine samples collected from pregnant marmoset mothers and then observed their offspring's play behavior as juveniles (5-10 months of age). In contrast to findings in rhesus macaques, hierarchical regression analyses showed that higher gestational testosterone levels, primarily in the second semester, were associated with decreased rough-and-tumble play in juveniles, and this relationship appears to be driven more so by males than females. We found no reliable associations between gestational androstenedione and juvenile play behavior. Our findings provide evidence to suggest that normative variation in levels of maternal androgen during gestation may influence developmental behavioral trajectories in marmosets in a way that contradicts previous findings in Old World primates.     

The type of DUOX-dependent ROS production is dictated by defined sequences in DUOXA

A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.