Metabolism of furazolidone by milk xanthine oxidase and rat liver 9000g supernatant: formation of a unique nitrofuran metabolite and an aminofuran derivative

In vitro metabolism of furazolidone (N-(5-nitro-2-furfuryliden)-3-amino-2-oxazolidone) was investigated by using milk xanthine oxidase and rat liver 9000g supernatant. As a result, a new type of reduction product was isolated as one of the main metabolites from the incubation mixture and it was tentatively identified as 2,3-dihydro-3-cyanomethyl-2-hydroxyl-5-nitro-1a, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3- b]furan. In addition, the present study demonstrated the formation of N-(5-amino-2-furfurylidene)-3-amino-2-oxazolidone as a minor metabolite of nitrofuran in a milk xanthine oxidase system. The aminofuran derivative was easily degraded by milk xanthine oxidase under aerobic, but not anaerobic, conditions. The degradation appears to be due to superoxide anion radicals, hydroxyl radicals, and/or singlet oxygen, which are produced in this enzyme system.     

Phospholipids and a novel glycine-containing lipoamino acid in Cytophaga johnsonae Stanier strain C21.

Two-dimensional thin-layer chromatography revealed that Cytophaga johnsonae contains at least 10 kinds of lipid, 2 of which are phospholipids, namely, phosphatidylethanolamine and phosphatidylcholine. One of the remaining lipids is a novel lipid that contains an amino acid. The structure of this unusual lipid (lipoamino acid) was resolved by chemical and physicochemical methods. The fatty acyl moiety of this lipid was diverse. The structure of the major molecular species of the lipid was determined as iso-3-hydroxy heptadecanoic acid, amide linked to glycine and esterified to isopentadecanoic acid. This type of glycine-containing lipid is a novel biological material which we have called cytolipin, basing this nomenclature on the genus of the bacterium. This is the first report of the lipid composition of C. johnsonae.     

Mutagenicity of pyridine- and quinoline-carbohydroxamic acid derivatives

11 pyridine- and 6 quinoline-carbohydroxamic acids were tested for mutagenicity on Salmonella typhimurium TA100 and TA98. The results are compared with those obtained for benzohydroxamic acid and 4 naphthohydroxamic acids. Most of them were mutagenic on both these tester strains. Of the pyridine derivatives, pyridine-2-carbohydroxamic acid was the most potent mutagen. Quaternarization of the pyridine-ring nitrogen prevented the induction of mutation to a marked extent. Among the quinoline derivatives, quinoline-6-carbohydroxamic acid showed potent mutagenicity similar to that of 2-naphthohydroxamic acid. The present study supports the proposal made previously that the mechanism for mutagenicity of hydroxamic acids involves Lossen rearrangement of the acid conjugates produced by enzymic acylation (or perhaps phosphorylation or sulfation) of the hydroxamic acids, followed by carbamoylation of the target molecule in the cell by the resultant isocyanate. The multiplicity of factors determining the mutagenic potency of hydroxamic acids is discussed.     

Cutting edge: The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks inflammatory injury in murine colitis

Inflammatory mediators such as TNF-alpha, IL-6, and IL-1 are important in the pathogenesis of inflammatory bowel diseases and are regulated by the activation of NF-kappaB. The aim of the present study was to investigate whether the NF-kappaB essential modulator (NEMO)-binding domain (NBD) peptide, which has been shown to block the association of NEMO with the IkappaB kinasebeta subunit (IKKbeta) and inhibit NF-kappaB activity, reduces inflammatory injury in mice with colitis. Two colitis models were established by the following: 1) inclusion of dextran sulfate sodium salt (DSS) in the drinking water of the mice; and 2) a trinitrobenzene sulfonic acid enema. Marked NF-kappaB activation and expression of proinflammatory cytokines were observed in colonic tissues. The NBD peptide ameliorated colonic inflammatory injury through the down-regulation of proinflammatory cytokines mediated by NF-kappaB inhibition in both models. These results indicate that an IKKbeta-targeted NF-kappaB blockade using the NBD peptide could be an attractive therapeutic approach for inflammatory bowel disease.     

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4′-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4′-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.     

Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3

Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln⁸⁶, Gly⁸⁷, Ile⁹¹, Thr⁹³, Trp⁹⁴, Ser¹⁶⁸, and Asn¹⁷³ are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.