Superoxide scavenging activity of pirfenidone-iron complex

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide () scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.     

Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae

The Saccharomyces cerevisiae MNN4 gene, which is involved inmannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide,has been cloned from a lambda phage containing a yeast chromosomeXI DNA fragment The MNN4 ORF encodes a protein of 1178 aminoacids. The deduced amino acid sequence shows a topology of typeII membrane proteins and has a unique repeated sequence of lysineand glutamic acid at the C-terminus. Disruption and overexpressionof MNN4 led to a decrease and increase, respectively, of themannosylphosphate content in cell wall mannans prepared fromboth mnn4 and wild type strains. A dramatic decrease of mannosylphosphateoccurs in     

Interleukin-6 Deficiency Does Not Affect Motor Neuron Disease Caused by Superoxide Dismutase 1 Mutation

MethodsMice with mutant SOD1 (G93A) transgene, a model for familial ALS, were used in this study. The expression of the major inflammatory cytokines, IL-6, IL-1β and TNF-α, in spinal cords of these SOD1 transgenic (TG) mice were assessed by real time PCR. Mice were then crossed with IL-6(-/-) mice to generate SOD1TG/IL-6(-/-) mice. SOD1 TG/IL-6(-/-) mice (n = 17) were compared with SOD1 TG/IL-6(+/-) mice (n = 18), SOD1 TG/IL-6(+/+) mice (n = 11), WT mice (n = 15), IL-6(+/-) mice (n = 5) and IL-6(-/-) mice (n = 8), with respect to neurological disease severity score, body weight and the survival. We also histologically compared the motor neuron loss in lumber spinal cords and the atrophy of hamstring muscles between these mouse groups.ResultsLevels of IL-6, IL-1β and TNF-α in spinal cords of SOD1 TG mice was increased compared to WT mice. However, SOD1 TG/IL-6(-/-) mice exhibited weight loss, deterioration in motor function and shortened lifespan (167.55 ± 11.52 days), similarly to SOD1 TG /IL-6(+/+) mice (164.31±12.16 days). Motor neuron numbers and IL-1β and TNF-α levels in spinal cords were not significantly different in SOD1 TG /IL-6(-/-) mice and SOD1 TG /IL-6 (+/+) mice.ConclusionThese results provide compelling preclinical evidence indicating that IL-6 does not directly contribute to motor neuron disease caused by SOD1 mutations.     

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Summary The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [³H]lathosterol to [³H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells. This work was supported by National Institute of Health grants CA40294 and CA37589 to G. H. Sato and by a grant from RJR nabisco Inc. Editor's Statement These results help identify the defect in myeloma cells leading to cholesterol auxotrophy. The use of these cells in hybridoma derivation adds practical utility to a detailed appreciation of cholesterol metabolism in these cultures.     

Discovery of orally efficacious RORγt inverse agonists. Part 2: Design,synthesis, and biological evaluation of novel tetrahydroisoquinoline derivatives

A series of tetrahydroisoquinoline derivatives were designed, synthesized, and evaluated for their potential as novel orally efficacious retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of Th17-driven autoimmune diseases. We carried out cyclization of the phenylglycinamide core by structure-based drug design and successfully identified a tetrahydroisoquinoline carboxylic acid derivative 14 with good biochemical binding and cellular reporter activity. Interestingly, the combination of a carboxylic acid tether and a central fused bicyclic ring was crucial for optimizing PK properties, and the compound 14 showed significantly improved PK profile. Successive optimization of the carboxylate tether led to the discovery of compound 15 with increased inverse agonistic activity and an excellent PK profile. Oral treatment of mice with compound 15 robustly and dose-dependently inhibited IL-17A production in an IL23-induced gene expression assay.     

Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src

The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST–Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. K_m for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.