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Intraperitoneally administered chondroitin sulfate E inhibited the development of antibody-induced arthritis, a model of rheumatoid arthritis, while chondroitin 4-sulfate showed no effects. Chondroitin sulfate E inhibited in vitro differentiation of osteoclasts, which play key roles in the etiology of rheumatoid arthritis. One of the targets of chondroitin sulfate E is midkine, a heparin-binding growth factor or cytokine. Indeed, a chimeric-type siRNA for midkine inhibited the development of antibody-induced arthritis and adhesion of the omentum to the injured abdominal wall. These results indicate the significance of midkine as a molecular target to treat or prevent rheumatoid arthritis and adhesion after surgery, and the utility of chondroitin sulfate E to inhibit midkine in vivo.

In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH(2)Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB(2), and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4x10(8)/ml) were preincubated with drugs for 5min at 37 degrees C prior to the incubation with AA (40microM) for 2min at 37 degrees C. HOCl (50-250microM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5-67% inhibition) and COX metabolites (TXB(2), 33-73% inhibition; 12-HHT, 27-74% inhibition). Although Tau-Cl and Glu-Cl up to 100microM were without effect on the formation of 12-HETE, TXB(2) and 12-HTT, NH(2)Cl showed a strong inhibition on the formation of all three metabolites (10-100microM NH(2)Cl, 12-HETE, 21-92% inhibition; TXB(2), 58-94% inhibition; 12-HHT, 36-92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH(2)Cl, and taurine restoring that induced by both NH(2)Cl and HOCl. These results suggest that NH(2)Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH(2)Cl-induced alterations in the COX and LOX pathways in vivo.

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Midkine (MK) is a heparin-binding growth factor, which promotes growth, migration, and survival of various cells, and MK expression is increased in many human carcinomas. We determined the urinary MK level by enzyme-linked immunoassay. Taking 311pg/mg creatinine as a cut-off level, 70% of patients with various carcinomas (n=142) gave positive values, while only 5.5% of healthy volunteers (n=330) did. In case of gastric carcinoma, 17 out of 21 patients with stage 1 tumor were positive. Urinary MK levels are expected to become a convenient marker as an aid in detection of tumors.

Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK., (Copyright 2000 Academic Press.)

Effect of peroxynitrite (ONOO-) on the conversion of xanthine dehydrogenase to oxidase in rabbit liver was examined. ONOO- (25-200 microM) induced the conversion of xanthine dehydrogenase to oxidase in a dose-dependent manner. The addition of hydroxyl radical scavengers (mannitol and dimethyl sulfoxide) gave no alteration in the ONOO(-)-induced conversion of xanthine dehydrogenase to oxidase, implying that the action of ONOO- is not due to hydroxyl radicals which may be formed from ONOO-. The experiment utilizing dithiothreitol also revealed that the action of ONOO- might be due to oxidation of sulfhydryl group of xanthine dehydrogenase. These results suggest that ONOO- has the potential to convert xanthine dehydrogenase to oxidase, and that this effect may be correlated with cytotoxic actions of ONOO-.

Tacrolimus(FK506) is a strong immuno-suppressant and shows its activity through inhibiting IL-2 mRNA transcription by forming pentameric complex with intracellular receptor(FK506 binding protein 12 kDa or FKBP12), Ca2+, calmodulin, and calcineurin. Here, we report the binding activity to FKBP12, the pentameric complex formation and Con-A response inhibiting activities of 7 metabolites. C15-demethylated metabolite(M-3) needed higher quantity to compete in Con-A assay and in pentamer formation assay, although it binds more strongly to FKBP12. The result suggests that the ability to form a pentameric complex is not a two step reaction with the first binding to FKBP12, but a single step reaction by components for the pentamer formation.