4,4'-Diarylsulfanyl-2,2',5,5'-tetraoxybiaryl derivatives as a water-soluble fluorescent dye.

4,4'-Disulfanyl-2,2',5,5'-tetrahydrobiaryl (5,5'-disulfanyl hydroquinone dimer) derivatives were readily synthesized from benzoquinone and thiols via an oxidative coupling reaction. The hydroquinone dimers showed strong fluorescence upon excitation at 330 nm, and it was observed that the presence of the sulfanyl groups at the C4 and C4' positions is important for achieving strong photoluminescence. The tetrapotassium salts of the hydroquinone dimers also showed good water solubility, but the fluorescence disappeared rapidly on dissolution in water. 2,2'- and 5,5'-protected biaryls were synthesized. The dipotassium salt of the 5,5'-dimethoxy-2,2'-dihydroxy derivative was observed to show good and stable fluorescence in water, while the dipotassium salt of the 2,2'-dimethoxy-5,5'-dihydroxy derivative showed less water solubility. Introduction of propargyl groups was demonstrated to provide a convenient method for installing amino acids derivatives. This derivatization afforded potentially useful compounds for attaching the biologically active fragment to the fluorescent unit.

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli.

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81mM (25g/L) NeuAc in 22h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22h reaction with 540mM (120g/L) GlcNAc in a 5L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172mM (53g/L) NeuAc.

Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody.

Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.

S-adenosyl-L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine gamma-synthase gene in Arabidopsis.

Cystathionine gamma-synthase, the first committed enzyme of methionine biosynthesis in higher plants, is encoded by the CGS1 gene in Arabidopsis thaliana. We have shown previously that the stability of the CGS1 mRNA is negatively regulated in response to methionine application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M. & Naito, S. (1999) Science 286, 1371-1374]. To determine whether methionine itself is the effector of the CGS1 exon 1-mediated posttranscriptional regulation, we carried out transfection experiments. The results suggested that, rather than methionine, S-adenosyl-L-methionine (AdoMet), or one of its metabolites, acts as the effector of this regulation. To further identify the actual effector, we exploited the wheat germ in vitro translation system. The effects of various metabolites and analogs of AdoMet were tested by using RNA carrying a CGS1 exon 1-reporter fusion. These tests identified AdoMet as the effector of this regulation. S-adenosyl-L-ethionine, an analog of AdoMet, also had effector activity. A. thaliana mto1 mutants, which are deficient in this regulation, showed a much reduced response to AdoMet in vitro, with a leaky allele showing a less reduced response. RNA translated in vitro in the presence of AdoMet contained a 5'-truncated RNA species, similar to the one that we previously suggested was an in vivo degradation intermediate of CGS1 mRNA. Together, the results show that the basic reactions of CGS1 exon 1-mediated posttranscriptional regulation occur in the wheat germ in vitro translation system, and that AdoMet acts as the effector.