Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-kappaB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.     

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay.

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.     

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

KIR,HLA, and IL28B Variant Predict Response to Antiviral Therapy in Genotype 1 Chronic Hepatitis C Patients in Japan

Natural killer cell responses play a crucial role in virus clearance by the innate immune system. Although the killer immunoglobulin-like receptor (KIR) in combination with its cognate human leukocyte antigen (HLA) ligand, especially KIR2DL3-HLA-C1, is associated with both treatment-induced and spontaneous clearance of hepatitis C virus (HCV) infection in Caucasians, these innate immunity genes have not been fully clarified in Japanese patients. We therefore investigated 16 KIR genotypes along with HLA-B and -C ligands and a genetic variant of interleukin (IL) 28B (rs8099917) in 115 chronic hepatitis C genotype 1 patients who underwent pegylated-interferon-α2b (PEG-IFN) and ribavirin therapy. HLA-Bw4 was significantly associated with a sustained virological response (SVR) to treatment (P = 0.017; odds ratio [OR] = 2.50, ), as was the centromeric A/A haplotype of KIR (P = 0.015; OR 3.37). In contrast, SVR rates were significantly decreased in patients with KIR2DL2 or KIR2DS2 (P = 0.015; OR = 0.30, and P = 0.025; OR = 0.32, respectively). Multivariate logistic regression analysis subsequently identified the IL28B TT genotype (P = 0.00009; OR = 6.87, 95% confidence interval [CI] = 2.62 - 18.01), KIR2DL2/HLA-C1 (P = 0.014; OR = 0.24, 95% CI = 0.08 - 0.75), KIR3DL1/HLA-Bw4 (P = 0.008, OR = 3.32, 95% CI = 1.37 - 8.05), and white blood cell count at baseline (P = 0.009; OR = 3.32, 95% CI = 1.35 - 8.16) as independent predictive factors of an SVR. We observed a significant association between the combination of IL28B TT genotype and KIR3DL1-HLA-Bw4 in responders (P = 0.0019), whereas IL28B TT along with KIR2DL2-HLA-C1 was related to a non-response (P = 0.0067). In conclusion, combinations of KIR3DL1/HLA-Bw4, KIR2DL2/HLA-C1, and a genetic variant of the IL28B gene are predictive of the response to PEG-IFN and ribavirin therapy in Japanese patients infected with genotype 1b HCV.     

Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

Y-40138, a multiple cytokine production modulator, protects against D-galactosamine and lipopolysaccharide-induced hepatitis

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs are associated with tumor necrosis factor (TNF) production. D-Galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure. In this model, TNF-alpha plays a central role in the pathogenesis of D-GalN/LPS-induced liver injury in mice. Y-40138, N-[1-(4-[4-(pyrimidin-2-yl)piperazin-1-yl]methyl phenyl)cyclopropyl] acetamide.HCl inhibits TNF-alpha and augments interleukin (IL)-10 production in LPS-injected mice in plasma. In the present study, we examined the effect of Y-40138 on D-GalN/LPS-induced hepatitis. Y-40138 (10mg/kg, i.v.) significantly suppressed TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) production and augmented IL-10 production in plasma. In addition, Y-40138 significantly inhibited TNF-alpha production induced by direct interaction between human T lymphocytes and macrophages. Y-40138 suppressed plasma alanine transaminase (ALT) elevation and improved survival rate in D-GalN/LPS-injected mice, and it is suggested that the protective effect of Y-40138 on hepatitis may be mediated by inhibition of TNF-alpha and MCP-1, and/or augmentation of IL-10. This compound is expected to be a new candidate for treatment of cytokine and/or chemokine-related liver diseases such as alcoholic hepatitis.     

HTLV-I and leukemogenesis

Human T-cell leukemia virus type I (HTLV-I) is a causative virus of adult T-cell leukemia (ATL). ATL is a highly aggressive neoplastic disease of CD4 positive T lymphocyte, which is featured by the pleomorphic tumor cells with hypersegmented nuclei, called " flower cell". HTLV-I increases its copy number by clonal proliferation of the host cells, not by replication of the virus. Therefore, HTLV-I eventually induces ATL. Tax, encoded by HTLV-I pX region, has been recognized as a protein that plays a central role of the transformation of HTLV-I-infected cells by its pleiotropic actions. However, fresh ATL cells frequently lose Tax protein expression by several mechanisms. Recently, HBZ was identified in the complementary strand of HTLV-I and it is suggested that HBZ is a critical gene in leukemogenesis. Furthermore, there is a long latency period before onset of ATL, indicating the multistep mechanisms of leukemogenesis. Therefore, it is suggested that multiple factors, such as viral proteins, genetic and epigenetic changes of host genome, and immune status of the hosts, could be implicated in leukemogenesis of ATL.     

Functional alterations induced by fusarium T-2 toxin in Madin-Darby bovine Kidney (MDBK) and primary fetal bovine Kidney (PFBK) cultures

We recently reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. The present report examined the influence of T-2 on selected biochemical parameters of these two culture types. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K⁺- dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10^?5M.