Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion

N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe(2+)-H(2)O(2) (Fenton's reagent) with Cu(2+) activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe(2+)-Cu(2+)-H(2)O(2) on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe(2+), Cu(2+), H(2)O(2) and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The (1)H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe(2+)-Cu(2+)-H(2)O(2)-NO.     

Enantioselectivity of Enzymatic Cleavage Reaction of 13-Hydroperoxylinolenic Acid to C6-Aldehyde and C12-Oxo Acid in Tea Chloroplasts

A protease with strict specificity to lysyl peptide bonds like that of Achromobacter protease I was purified from a crude enzyme powder obtained from a culture filtrate of Achromobacter lyticus M497-1 and characterized. The purified enzyme had the following differences from protease I. The enzyme had an isoelectric point of 5.3, lower than the value of 6.9 for protease I. The amino acid composition of the enzyme had higher proportions of His, Glu, and Gly and lower proportions of Arg and Thr than protease I. The enzyme was unstable (30% residual activity) in the presence of 7 m urea (pH 8.0, 30°C, 20 min); protease I was resistant to the same conditions (80% residual activity). The k_cat/Km values for the hydrolysis of Tos-Lys-OMe and Lys-pNA by the enzyme were lower than those of protease I.     

Synthesis of 3Z, 6Z-Dienoic Acids

3Z,6Z-Dienoic acids (C₈-C₁₂ and C₁₈) were for the first time synthesized by coupling 2-acetylenic bromides and 2-(3′-butynyloxy)-tetrahydropyrane followed by stereoselective hydrogenation and oxidation.     

Blätteralkohol (XIV) Synthese des 2-Propyl-5-äthyl-benzylalkohols

2-Propyl-5-ethyl-benzylalcohol (IX) was synthesized by an unequivocal route from propylbenzene, thereby establishing the previous deduction tentatively assigned to the leaf alcohol reaction product.¹⁾ This benzyl alcohol surmises one of a lemon-like flavor characteristic of manufactured black tea and an attempted search for this compound in the essential oil obtained by steamdistillation of manufactured black tea was made, but its existence has not so far been confirmed with a neutral fraction examined.     

Simultaneous ultrastructural identification of growth hormone and its messenger ribonucleic acid using combined immunohistochemistry and non-radioisotopicin situ hybridization: A technical note

Summary The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopicalin situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.