In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

Measurement of hydroxyl radical (*OH) in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because generation of *OH is believed to be one of the major triggers of radiation injury. In this study, *OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of *OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of alpha-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN-methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of *OH but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was *OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of *OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.     

Preparation and structural analysis of actinidain-processed atelocollagen of yellowfin tuna (Thunnus albacares)

Pepsin-hydrolyzed collagen (atelocollagen) is a trimer, consisting of alpha 1 and alpha 2 monomers, and shows molecular species corresponding to a monomer, dimer (beta chain), and trimer (gamma chain) by SDS-polyacrylamide gel electrophoresis. Atelocollagen was purified from yellowfin tuna (Thunnus albacares) by salt precipitation and cation-exchange chromatography. Enzymatic hydrolysis of the atelocollagen by actinidain, a cysteine protease purified from kiwifruit, was analyzed by SDS-polyacrylamide gel electrophoresis. The triple helical structure unique to collagen was retained in the atelocollagen as judged by circular dichroism spectra. The actinidain-processed atelocollagen showed only monomeric alpha 1 and alpha 2 chains, with no beta and gamma chains, by SDS-polyacrylamide gel electrophoresis; nevertheless, it retained the typical triple helical structure. It is suggested that actinidain cleaved the atelocollagen molecule at specific sites on the inside of the inter-strand cross-linking peptides.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

Essential role for the p40 subunit of interleukin-12 in neutrophil-mediated early host defense against pulmonary infection with Streptococcus pneumoniae: involvement of interferon-gamma

Interleukin (IL)-12 is a critical cytokine in the T helper (Th)1 response and host defense against intracellular microorganisms, while its role in host resistance to extracellular bacteria remains elusive. In the present study, we elucidated the role of IL-12 in the early-phase host defense against acute pulmonary infection with Streptococcus pneumoniae, a typical extracellular bacterium, using IL-12p40 gene-disrupted (IL-12p40KO) mice. IL-12p40KO mice were highly susceptible to S. pneumoniae infection, as indicated by the shortened survival time, which was completely restored by the replacement therapy with recombinant (r) IL-12, and increased bacterial counts in the lung. In these mice, recruitment of neutrophils in the lung was significantly attenuated when compared to that in wild-type (WT) mice, which correlated well with the reduced production of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF)-alpha in the infected tissues at the early phase of infection. In vitro synthesis of both cytokines by S. pneumoniae-stimulated lung leukocytes was significantly lower in IL-12p40KO mice than in WT mice, and addition of rIL-12 or interferon (IFN)-gamma restored the reduced production of MIP-2 and TNF-alpha in IL-12p40KO mice. Neutralizing anti-IFN-gamma monoclonal antibody (mAb) significantly decreased the effect of rIL-12. Anti-IFN-gamma mAb shortened the survival time of infected mice and reduced the recruitment of neutrophils and production of MIP-2 and TNF-alpha in the lungs. Our results indicated that IL-12p40 plays a critical role in the early-phase host defense against S. pneumoniae infection by promoting the recruitment of neutrophils to the infected tissues.     

The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.