The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.     

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.     

Anti-monocyte chemoattractant protein-1 gene therapy inhibits vascular remodeling in rats: blockade of MCP-1 activity after intramuscular transfer of a mutant gene inhibits vascular remodeling induced by chronic blockade of NO synthesis.

Monocyte chemoattractant protein-1 (MCP-1) may play an essential part in the formation of arteriosclerosis by recruiting monocytes into the arterial wall. Thus, we devised a new strategy for anti-MCP-1 gene therapy against arteriosclerosis by transfecting an amino-terminal deletion mutant (missing the amino-terminal amino acids 2 to 8) of the human MCP-1 gene into a remote organ (skeletal muscles). Intramuscular transduction with the mutant MCP-1 gene blocked monocyte recruitment induced by a subcutaneous injection of recombinant MCP-1. In a rat model in which the chronic inhibition of endothelial nitric oxide synthesis induces early vascular inflammation as well as subsequent coronary vascular remodeling, this strategy suppressed monocyte recruitment into the coronary vessels and the development of vascular medial thickening, but did not reduce perivascular fibrosis. Thus, MCP-1 is necessary for the development of medial thickening but not for fibrosis in this model. This new strategy may be a useful and feasible gene therapy against arteriosclerosis.     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

Relative contribution of CD2 and LFA-1 to murine T and natural killer cell functions.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.     

A murine very late activation antigen-like extracellular matrix receptor involved in CD2- and lymphocyte function-associated antigen-1-independent killer-target cell interaction

CD2 and lymphocyte function-associated antigen (LFA)-1 are well known as T cell adhesion molecules involved in killer-target cell interactions. However, our recent study revealed that molecule(s) other than CD2 and LFA-1 might be involved in the lymphokine-activated killer (LAK) cell cytotoxicity against certain target cells. In order to characterize such unknown molecules, we established a mAb (RMV-7) which could inhibit CD2/LFA-1-independent LAK cell cytotoxicity and binding to target cells at the effector site. The Ag identified by RMV-7 appeared on splenic T cells late after mitogenic stimulation and was a noncovalently linked heterodimer composed of a 140-kDa alpha-chain and a 95-kDa beta-chain. RMV-7 blocked LAK cell binding to fibronectin (FN), fibrinogen, and vitronectin but not that to laminin or type IV collagen, indicating that the RMV-7-defined molecule is a unique extracellular matrix receptor for FN, fibrinogen, and vitronectin. One of its ligand, FN, was found on the surface of several target cells, and LAK cell cytotoxicity against them was blocked by anti-FN antibody at the target site. Similarly, cytotoxicity of a H-2d-specific CTL clone was inhibited by RMV-7 and anti-FN antibody as well. These results indicate that a unique very late activation Ag-like extracellular matrix receptor on murine CTL and LAK cells contributes to target cell binding and cytotoxicity.