Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.     

Genomic sequence and organization of the family of CNR/Pcdhalpha genes in rat

CNR/Pcdhalpha family proteins are known as synaptic cadherins and Reelin receptors. Here we report the complete genomic sequence and organization of the rat CNR. The rat CNR cluster encodes 15 variable and 3 constant exons. The genomic organizations of the rat, mouse, and human CNR/Pcdhalpha are orthologous. The percentage identity of the coding regions between the rat and the mouse is 93.6% on average at the nucleic acid level, and between rat and human it is 82.8%. The rat CNRs (v1-v13) also contain an RGD motif in the extracellular cadherin 1 domains and cysteine repeats that are characteristic of the transmembrane and cytoplasmic domains of CNR proteins. The number of variable exons in the rat CNR cluster is identical to that of the human. The rat CNR cluster has one more variable exon than is found in laboratory mouse strains, because in the mouse a variable exon located between v7 and v8 is divided by the insertion of a retrotransposon. This exon is not disrupted in the rat, in which it is transcribed. By in silico analysis, CNR/Pcdhalpha was also mapped to rat chromosome 18, but the orientation was opposite for the mouse CNR/Pcdhalpha gene cluster. The relative expression profiles of the rat CNRs (v1-v13) show that all the CNRs are transcribed, but there are variations in the expression ratios among the CNRs.     

Current LCA database development in Japan -results of the LCA project

In 1998, the Japan’s Ministry of Economy, Trade, and Industry (METI) launched a five-year national project entitled ‘Development of Life Cycle Impact Assessment for Products’ (commonly known as ‘the LCA Project’). The purpose of the project is to develop common LCA methodology as well as a highly reliable database that can be shared in Japan. Activities over these five years have resulted in the supply of LCI data on some 250 products. Industrial associations voluntarily provided data. The results of these activities are currently being made available on the Internet on a trial basis in the form of an LCA database. In addition, a method entitled ‘Life-cycle Impact assessment Method based on Endpoint modeling (LIME)’ was developed. It is expected that these results will be widely used in Japan in the future. This paper presents an outline of the results of the research and development that has been conducted in the LCA Project in Japan.     

Development of an efficient enzymatic production of gamma-D-glutamyl-L-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial gamma-glutamyltranspeptidase

Gamma-D-Glutamyl-L-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial. Because gamma-D-glutamyl-L-tryptophan has several reactive groups in its molecule, consists of D- and L-amino acids, and is connected by gamma-glutamyl linkage, its chemical synthesis is complicated. An efficient enzymatic method to synthesize gamma-D-glutamyl-L-tryptophan from D-glutamine and L-tryptophan employing bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 50 mM D-glutamine, 50 mM L-tryptophan, and 0.2 U ml(-1) gamma-glutamyltranspeptidase, pH 9-9.5, and incubation at 37 degrees C for 5 h. After a 5 h incubation, 33 mM gamma-D-glutamyl-L-tryptophan was obtained, the conversion rate being 66%. The product was purified by Dowex 1 x 8 column and was considered to be gamma-D-glutamyl-L-tryptophan.     

Thienopyridine and benzofuran derivatives as potent anti-tumor agents possessing different structure-activity relationships

(3-Amino-6-thiophen-2-yl-thieno[2,3-b]pyridin-2-yl)phenylmethanone (3) was discovered as a new type of cytotoxic agent selective against a tumorigenic cell line. The molecular structure of a previously reported compound, (4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)phenylmethanone (2), had remarkably similar bioisosteric substructures to that of compound 3. Although the relationship between the molecular structure and biological activity of each derivative synthesized from these two hit compounds (2 and 3) were studied, unexpectedly no correlation was observed. However, after further synthetic study from 3, one of the most potent derivative (10k) having a different SAR profile from 2, was discovered.