A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.     

Histamine release from rat mast cells by Vibrio vulnificus protease

Abstract Vibrio vulnificus protease (VVP) stimulated histamine release from isolated mast cells in a dose- and temperature-dependent manner within a range of 0.2–4.0 μ g/0.5 ml. Histamine release was accompanied by degranulation, and no leakage of lactate dehydrogenase from cells was observed, indicating that the histamine release was not due to cytolysis but to exocytosis. This release, completed within 30 s at 37°C, suggested that the mechanism of action of VVP on mast cells is different from that of other proteases, such as trypsin or α-chymotrypsin, which release histamine from the cells slowly.     

14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels

Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.     

A practical approach to N-glycan production by hydrazinolysis using hydrazine monohydrate

Hydrazinolysis is a versatile method to liberate N-linked glycans from glycoproteins. However, the method is usually performed with anhydrous hydrazine, a highly toxic and explosive chemical used in rocket fuel. Thus despite the need to produce functionally important glyco-materials, hydrazinolysis is limited to small scale (e.g., 0.2-1 mL) reactions. In the present study, we report an alternative procedure for hydrazinolysis using hydrazine monohydrate in place of anhydrous hydrazine. The developed procedure was applied to both purified glycoproteins (Taka-amylase and transferrin) and hen egg yolk protein fraction with comparable yields to the traditional method using anhydrous hydrazine. The sialyl linkage of alpha2-6disialobiantennary oligosaccharides proved to be fully stable. The developed procedure facilitated the large-scale preparation of N-linked glycans. The new method should make a substantial contribution to both small- and large-scale production of functional glycans, including therapeutically relevant human-type glycans.     

Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys(6)- and Cys(111)-disulfide cross-linked form and targeted it for proteasomal degradation.     

Chemopreventive effects of hydroxymatairesinol on uterine carcinogenesis in Donryu rats

Hydroxymatairesinol (HMR), obtained from the heartwood of spruce (Picea abies), has been demonstrated to exert chemo-preventive effects on the development of mammary tumors in rats. To examine the influence of HMR on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 11 weeks of age and fed thereafter 0, 200, or 600 ppm HMR mixed in the soy-containing diet until 15 months of age. Incidences of uterine adenocarcinoma in both 200 and 600 ppm HMR-dosed groups were significantly reduced to 11% and 15%, respectively, less than 50% of 0 ppm, at the end of the experiment (P < 0.05). A delay in the start of persistent estrus by HMR was observed at 8 months of age compared with controls given carcinogen alone. From urinalysis, HMR was metabolized mainly to enterolactone and hydroxyenterolactone. These findings suggest that HMR or its metabolites exert chemo-preventive effects in the rat ENNG-uterine carcinogenesis model.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tsukushi cooperates with VG1 to induce primitive streak and Hensen's node formation in the chick embryo

Three classes of signaling molecule, VG1, WNT and BMP, play crucial roles in axis formation in the chick embryo. Although VG1 and WNT signals have a pivotal function in inducing the primitive streak and Hensen's node in the embryo midline, their action is complemented by that of BMP antagonists that protect the prospective axial tissue from the inhibitory influence of BMPs secreted from the periphery. We have previously reported that a secreted factor, chick Tsukushi (TSK), is expressed in the primitive streak and Hensen's node, where it works as a BMP antagonist. Here, we describe a new crucial function for TSK in promoting formation of the primitive streak and Hensen's node by positively regulating VG1 activity. We provide evidence that TSK directly binds VG1 in vitro, and that TSK and VG1 functionally interact in axis formation, as shown by biological assays performed in chick and Xenopus embryos. Furthermore, we show that alternative splicing of TSK RNA leads to the formation of two isoforms (TSKA, originally designated as TSK, and TSKB) that differ in their C-terminal region. Biochemical and biological assays indicate that TSKB is a much weaker BMP antagonist than TSKA, although both isoforms efficiently interact with VG1. Remarkably, although both TSKA and TSKB are expressed throughout the early extending primitive streak, their expression patterns diverge during gastrulation. TSKA expression concentrates in Hensen's node, a well-known source of anti-BMP signals, whereas TSKB accumulates in the middle primitive streak (MPS), a region known to work as a node-inducing center where VG1 expression is also specifically localized. Loss-of-function experiments demonstrate that TSKB, but not TSKA, function is required in the MPS for induction of Hensen's node. Taken together, these results indicate that TSK isoforms play a crucial role in chick axis formation by locally modulating VG1 and BMP activities during gastrulation.     

Glucosylation of Salicyl Alcohol by Gardenia jasminoides Cell Cultures

Cultured cells of Gardenia jasminoides produced both salicinand isosalicin from exogenously supplied salicyl alcohol. Theglucosylation activity of the cells was highest in the exponentialphase of growth and ca. 70% of the added substrate was convertedto the glucosides within 4 days. The rate of glucosylation wasalso dependent on the medium composition such as auxin and sucroseconcentrations. The ratio of salicin to isosalicin formed fromsalicyl alcohol was influenced by the growth stage of the culturedcells. Salicin was converted to isosalicin when exogenouslyadded to the culture. (Received October 11, 1985; Accepted March 10, 1986)