Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT.

Although the a-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent a-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent a-chymases have unique biological activity different from the chymases of other species.Keywords: mast cell protease(s); chymase; elastase; chymotrypsin; substrate specificity; site-directed mutagenesis; homology modeling.Chymase is a chymotrypsin-like serine protease expressed exclusively in mast cells (MCs), where the protease is stored within the secretary granules and released along with tryptase, heparin, and histamine in response to allergen challenge or other stimuli [1]. Although the physiological function of this protease is still unclear, it is probably involved in various allergic inflammatory reactions, cardiovascular diseases, and chronic inflammatory diseases [2]. For example, the proposed actions of chymase include induction of microvascular leakage [3], inflammatory cell accumulation [4], neutrophil and lymphocyte chemotaxis [5], stimulation of bronchial gland secretion [6], mast cell degranulation [7], extracellular matrix degradation [8][9][10][11][12][13], and cytokine metabolism [14][15][16][17].Based on phylogenetic analyses of a large set of cDNAderived sequences and comparison of the substrate preferences of a smaller set of purified enzymes, mammalian chymases have been divided into two families, the a-and b-chymase families [18,19]. Mice and rats have a number of chymase isozymes that belong to the a-chymase family (mouse mast cell protease-5/mMCP-5 and rat mast cell protease-5/rMCP-5) and the b-chymase family (mMCP-1, 2, 4, rMCP-1, 2, 4) [20,21]. Primates and dogs, on the other hand, are generally thought to have just a single a-chymase [22][23][24]. Across mammalian species, the primary structures of a-chymases are much more similar to each other than to those of the b-chymases. For example, the amino acid sequences for human chymase...

The acetylesterase (AE) activity of DVIM (diarrhea virus of infant mice) was assigned to the haemagglutinin-esterase (HE) protein. The substrate specificity was examined using the natural substrate bovine submaxillary mucin (BSM) and/or synthetic substrates p-nitrophenylacetate (p-NiA) and alpha-naphthylacetate (alpha-NA) and compared with several strains of MHV and influenza viruses. The AE of DVIM hydrolyzed the O-acetylester bond of BSM, and the two synthetic substrates p-NiA and alpha-NA in vitro. MHV-S reacted efficiently with both p-NiA and alpha-NA but less with BSM. Influenza virus (C/Miyagi/77) reacted with BSM efficiently, however reacted with p-NiA weakly, but not with alpha-NA at all. Thus, the AE-reactivity of DVIM was distinctly different from that of MHV-S and influenza C virus, suggesting that the AE of HE may have a modified function. Isolation of HE by the treatment with non ionic detergent NP40, resulted in globules approximately 5 nm in diameter. DVIM-binding proteins were demonstrated in the plasma membrane of mouse intestinal brush-border cells and hepatocytes. The same protein was recognized by MHV-S and MHV-4. The cell membranes obtained from these target tissues were substrates for the AE of DVIM. The biological importance of the HE protein for DVIM is discussed.

We prepared novel S-shaped liquid crystal oligomers, the 4,49-bis (v-(2-(v-(4-(cyanophenyl)phenyloxy)alkyloxy)phenyloxy)alkyloxybiphenyls (S-n), and investigated their transition behaviour. The oligomer with even-numbered spacers (S-6) exhibited a nematic phase, whereas the oligomer with odd-numbered spacers (S-7) exhibited nematic and smectic A phases. Transition properties of the S-shaped oligomers are compared with those of the corresponding monomeric and U-shaped dimeric compounds. The entropy changes associated with the I-N transition for the oligomers were found to have small values, similar to that of the monomeric compound. A biaxial smectic phase was induced below the SmA phase in some binary mixtures of S-6 and a phenylpyrimidine derivative (8-PYP-6O). On the other hand, the SmA phase was stabilized in the mixtures of S-7 and 8-PYP-6O. We discuss the transition behaivour of the S-shaped oligomers in terms of their molecular shape.

We analyzed the characteristics of seven monoclonal antibodies (mAbs) raised against purified HE (hemagglutinin-esterase) glycoprotein of the murine coronavirus DVIM (diarrhea virus of infant mice). Immunocrossreaction of these mAbs with JHM and/or MHV-S suggest that antigenic epitopes of HE of DVIM are similar to those of JHM and/or MHV-S. Four mAbs (1b4, 3a28, 4c19, 10b7), designated as group A mAbs, strongly inhibited both HA and AE activities. On the other hand, three mAbs (5a3, 6a6, 13a4), referred to as group B, had a comparatively weak HA inhibition activity. These results indicate that the antigenic epitopes of this glycoprotein can be classified into at least two groups and that the functional sites of HA and AE activities are similar but not identical. Neutralizing activity was shown in group A mAbs exclusively, suggesting that the ratio of HA and/or AE activities may play important roles in the cell fusion activity of DVIM-infected cells.