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.     

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.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.