Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (r Si -HLP). r Si -HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1β and TNF-α) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of r Si -HLP was heat-unstable. In subsequent studies, r Si -HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that r Si -HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si -HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.     

Design, synthesis and characterization of podocarpate derivatives as openers of BK channels

We found that the podocarpic acid structure provides a new scaffold for chemical modulators of large-conductance calcium-activated K(+) channels (BK channels). Structure-activity analysis indicates the importance of both the arrangement (i.e., location and orientation) of the carboxylic acid functionality of ring A and the hydrophobic region of ring C for expression of BK channel-opening activity.     

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

Screening for candidate genes involved in tolerance to organic solvents in yeast

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents.     

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

 A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25⁺, CD54⁺, CD69⁺ or CD71⁺ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression. Received: 5 June 1997 / Accepted: 12 September 1997