The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.     

Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.     

Allosteric switching by mutually exclusive folding of protein domains

Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.     

Distinct requirements for IL-7 in development of TCR gamma delta cells during fetal and adult life

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta cells.     

Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.     

Inducible costimulatory molecule-B7-related protein 1 interactions are important for the clonal expansion and B cell helper functions of naive,Th1, and Th2 T cells

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.     

A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation,interferes with early B cell development

Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.     

Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-alpha production in monocytes. Degradation of I-kappa B alpha suggested involvement of NF-kappa B activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-alpha and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients' DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.     

Estrogen receptor alpha mediates estrogen's immune protection in autoimmune disease

Estrogens are known to influence a variety of autoimmune diseases, but it is not known whether their actions are mediated through classic estrogen receptor alpha (ERalpha). The presence of a functional ER was demonstrated in secondary lymphoid tissues, then ERalpha expression was shown at both the RNA and protein levels in these tissues. Use of ERalpha knockout mice revealed that both the estrogen-induced disease protection and the estrogen-induced reduction in proinflammatory cytokines were dependent upon ERalpha in the prototypic Th1-mediated autoimmune disease experimental autoimmune encephalomyelitis. These findings are central to the design of selective ER modifiers which aim to target biologic responses in specific organ systems.     

Fenfluramine-induced serotonergic neurotoxicity in mice: lack of neuroprotection by inhibition/ablation of nNOS

Previous studies have implicated a role for nitric oxide (NO) and peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity. The present study was undertaken to investigate whether NO is involved in serotonergic neurotoxicity caused by fenfluramine. In the first experiment, the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 25 mg/kg x 4) on fenfluramine (25 mg/kg x 4)-induced serotonergic neurotoxicity in Swiss Webster mice was investigated. In the second experiment, the effect of fenfluramine (25 mg/kg x 4) on nNOS (-/-) and wild-type (WT) mice was investigated. Fenfluramine induced hypothermia in all three mouse strains, and 7-NI had no thermoregulatory effect. Selective depletion of 5-HT and 5-HT transporter binding sites in the striatum, frontal cortex and hippocampus in all three mouse strains was observed, with no evidence of dopaminergic neurotoxicity. In the first experiment, 7-NI did not attenuate serotonergic neurotoxicity in Swiss Webster mice. In the second experiment, nNOS(-/-) and WT mice were equally sensitive to serotonergic neurotoxicity. These findings suggest that NO and peroxynitrite do not mediate fenfluramine-induced serotonergic neurotoxicity, and that NO is a selective mediator of amphetamines-induced dopaminergic neurotoxicity.