Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Effects of the soluble fibre pectin on intestinal cell proliferation, fecal short chain fatty acid production and microbial population.

AIM: Although pectin, a dietary fibre, has been suggested to possess some trophic effects on the intestine, the mechanisms involved remain unclear. This study aimed to evaluate the effects of pectin on rat intestinal cell proliferation and the intraluminal environment. METHODS: Control and pectin-fed rats were given a fibre-free elemental diet (ED) and an ED containing 2.5% pectin, respectively. On the 15th day, the length, weight and number of Ki-67-positive cells from each intestinal segment, and the short chain fatty acids (SCFAs) and microbial population in the caecum were measured. Plasma glucagon-like peptide-2 (GLP-2) concentration and GLP-2 receptor (GLP-2R) mRNA levels in the epithelium were also determined. RESULTS: Pectin supplementation resulted in significant increases in the length, weight, and number of Ki-67-positive cells in the ileum, caecum and colon. Although pectin supplementation did not affect the caecal microbial flora that produced SCFAs, the caecal SCFA content was significantly increased. Pectin supplementation also induced an increase in the plasma GLP-2 concentration, but did not affect the GLP-2R mRNA levels in the small intestine. CONCLUSIONS: The increases in the caecal SCFAs and plasma GLP-2 levels induced by pectin supplementation may cause mucosal proliferation in the lower intestinal tract.

Metal ion-catalyzed Diels-Alder and hydride transfer reactions. Catalysis of metal ions in the electron-transfer step.

Rates of Diels-Alder cycloaddition of anthracenes with p-benzoquinone and its derivatives as well as rates of hydride-transfer reactions from 10-methyl-9,10-dihydroacridine to the same series of p-benzoquinones are accelerated significantly in the presence of metal ions in acetonitrile. An extensive comparison of the catalytic effects of metal ions in electron transfer from one-electron reductants (cobalt tetraphenylporphyrin and decamethylferrocene) to p-benzoquinones with those in the Diels-Alder reactions of the quinones as well as the hydride-transfer reactions has revealed that the catalysis of metal ions in each case is ascribed to the 1:1 and 1:2 complexes formed between the corresponding semiquinone radical anions and metal ions. The transient absorption and ESR spectra of the semiquinone radical anion-metal ion complexes are detected directly in the electron-transfer reduction of p-benzoquinone derivatives in the presence of metal ions. The catalytic reactivities of a variety of metal ions in each reaction are well correlated with the energy splitting values of pi(g) levels because of the complex formation between O(2)(.-) and M(n+), which are derived from the g(zz) values of the ESR spectra of the O(2)(.-)-M(n+) complex.