Search

Hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond clevage. Changes at position 8 caused variation in the A -1sugar pucker, A8 and U8 appeared to represent nonproductive in which their 2' - OH group mimicked the pro- R, nonbridging oxygen of the vandate transition-state complex.

Crystal structures of minimal, junctionless all-RNA hairpin ribozyme are solved. It is found that structures of the U39C and U39(C3) mutations appeared to shift the conformational equilibrium of U37 into the sequestered state while providing a structure-based rationale for their differing metal depending a structure-based rationale for their differing metal dependencies.

The crystal structure of ornithine cyclodeaminase in complex with NADH, refined to 1.80 angstroms resolution is described. Possible catalytic residues are described, along with the amino acid signature sequences that differentiate OCD from other members of the (mu)-crystallin family.

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes. [ABSTRACT FROM AUTHOR]

Influenza virus vaccination strategies are focused upon the elicitation of protective antibody responses through administration of viral protein through either inactivated virions or live attenuated virus. Often overlooked in this strategy is the CD4 T cell response: how it develops into memory, and how it may support future primary B cell responses to heterologous infection. Through the utilization of a peptide-priming regimen, this study describes a strategy for developing CD4 T cell memory with the capacity to robustly expand in the lung-draining lymph node after live influenza virus infection. Not only were frequencies of antigen-specific CD4 T cells enhanced, but these cells also supported an accelerated primary B cell response to influenza virusderived protein, evidenced by high anti-nucleoprotein (NP) serum antibody titers early, while there is still active viral replication ongoing in the lung. NP-specific antibody-secreting cells and heightened frequencies of germinal center B cells and follicular T helper cells were also readily detectable in the draining lymph node. Surprisingly, a boosted memory CD4 T cell response was not sufficient to provide intermolecular help for antibody responses. Our study demonstrates that CD4 T cell help is selective and limiting to the primary antibody response to influenza virus infection and that preemptive priming of CD4 T cell help can promote effective and rapid conversion of naive B cells to mature antibody-secreting cells. [ABSTRACT FROM AUTHOR]

Influenza-specific immunity in humans is unique because there are repeated exposures to viral strains containing genetically conserved epitopes recruiting memory CD4 T cells and novel epitopes stimulating naive CD4 T cells, possibly resulting in competition between memory and naive lymphocytes. In this study, we evaluated the effect of this competition on CD4 T cell and B cell response specificity using a murine model of sequential influenza infection. We found striking and selective decreases in CD4 T cell reactivity to nonconserved hemagglutinin (HA) epitopes following secondary influenza infection. Surprisingly, this shift in CD4 T cell specificity was associated with dramatic decreases in HA-specific Ab. These results suggest that repeated exposure to influenza viruses and vaccines containing conserved internal proteins may have unintended and negative consequences on the ability to induce HA-specific Ab to novel pandemic strains of influenza. These finding could have important implications on pandemic influenza preparedness strategies. [ABSTRACT FROM AUTHOR]

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses. [ABSTRACT FROM AUTHOR]

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood- brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke. [ABSTRACT FROM AUTHOR]

Ornithine cyclodeaminase (OCD) is a member of the μ-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia. In order to elucidate the functional groups of this enzyme that arc involved in catalysis. the crystallization of OCL) from Pseudomonas Putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc. (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified. Several conditions could he reproduced on a larger scale as vapor-diffusion experiments in-house. The best diffraction-quality crystals were obtained front solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 Å resolution. Crystals belonged to space group P212121, with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 Å. The VM was 2.1 ų Da-1. corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit. The structure determination is under way using experimental phasing methods. [ABSTRACT FROM AUTHOR]

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus. [ABSTRACT FROM AUTHOR]

Abstract: There are a number of related goals of influenza vaccination, including elicitation of protective antibodies and induction of cellular CD4 and CD8+ T cell responses. Because CD4+ T cell expansion and functionality are influenced by peptide specificity and T cell gene expression can be modified by repeated re-stimulations, it is important to evaluate how frequent influenza vaccinations affect CD4+ T cell dependent functions in protective immunity to influenza. Trivalent influenza vaccines (TIV) have production of neutralizing antibodies to HA as their primary goal and main criteria for efficacy. Accordingly, they are not characterized for any other viral components. In the current study, we evaluated whether other influenza virus proteins were present in commercial TIV at levels sufficient for immunogenicity in vivo. Mice that differed with regard to their expressed class II molecules were used in concert with peptide-stimulated cytokine ELISPOT assays to comprehensively evaluate the CD4+ T cell antigen specificity induced by the TIV. Our studies revealed that NA, NP, M1 and NS1 were present in sufficient quantities in the TIV to prime and boost CD4+ T cells. These results suggest that in humans, the broad CD4+ T cell repertoire induced by live infection is continually boosted and maintained throughout life by regular vaccination with licensed intramuscular split vaccines. The implications raised by our findings on CD4+ T cell functionality in influenza are discussed. [Copyright &y& Elsevier]

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.