A role of methionine 100 in facilitating PYP(M)-decay process in the photocycle of photoactive yellow protein

PYP (photoactive yellow protein) is a photoreceptor protein, which is activated upon photo-isomerization of the p-coumaric acid chromophore and is inactivated as the chromophore is thermally back-isomerized within a second (in PYP(M)-to-PYP(dark) conversion). Here we have examined the mechanism of the rapid thermal isomerization by analyzing mutant PYPs of Met100, which was previously shown to play a major role in facilitating the reaction [Devanathan, S. et al. (1998) Biochemistry 37, 11563-11568]. The mutation to Lys, Leu, Ala, or Glu decelerated the dark state recovery by one to three orders of magnitude. By evaluating temperature-dependence of the kinetics, it was found that the retardation resulted unequivocally from elevations of activation enthalpy (DeltaH( double dagger )) but not the other parameters such as activation entropy or heat capacity changes. Another effect exerted by the mutations was an up-shift of the apparent pK(a) of the chromophore [the pK(a) of a titratable group (X) that controls the pK(a) of the chromophore] in the PYP(M)-decay process. The pK(a) up-shift and the DeltaH( double dagger ) elevation show an approximately linear correlation. We, therefore, postulate that the role of Met100 is to reduce the energy barrier of the PYP(M)-decay process by an indirect interaction through X and that the process is thereby facilitated.     

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.     

Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Origin of two isoprenoid units in a lavandulyl moiety of sophoraflavanone G from Sophora flavescens cultured cells

Cell suspension cultures of Sophora flavescens produced large amounts of sophoraflavanone G, an 8-lavandulylated flavanone and lupalbigenin, a 6,3'-di-dimethylallylated isoflavone, by the simultaneous addition of cork tissues and methyl jasmonate. The labeling pattern of the isoprene units resulting after administration of [1-13C] glucose into the cell cultures in the presence of the above additives revealed that two isoprene units in the lavandulyl group of sophoraflavanone G and two dimethylallyl groups of lupalbigenin were biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway.     

Two novel oligosaccharides formed by 1F-fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.)

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-beta-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose, O-beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside and 1F(1-beta-D-fructofuranosyl)2-4G-beta-D-galactopyranosylsucrose, [O-beta-D-fructofuranosyl-(2-->1)]2-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcusfaecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.     

Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.     

Dietary magnesium increases calcium absorption of ovine small intestine in vivo and in vitro

The purpose of this study was to investigate whether or not an increase in dietary Mg intake increases Ca absorption in the ovine gastrointestinal tract. In an in vivo experiment, an increase in the infused MgCl2 level (0.0, 25.0 and 75.0 mg Mg x kg BW(-1) x day(-1) with 75.0 mg Ca x kg BW(-1) x day(-1) as CaCl2) into the rumen for ten days significantly decreased fecal excretion but increased urinary excretion (P < 0.05) of Ca in five castrated male sheep. Apparent Ca absorption tended to increase (P = 0.067) whilst the retention and plasma concentration of Ca were not changed. In an in vitro experiment with isolated segments from the rumen, upper jejunum, cecum and upper colon under the presence of an electrochemical gradient, the mucosal to serosal Ca flux rate was significantly greater in the presence of 60.0 mM as compared with 1.2 mM MgCl2 (P < 0.05). From these results, we conclude that the mucosal Mg has the ability to increase the Ca absorption in the gastrointestinal tract in sheep when the dietary Mg level is raised.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Radical scavenging activities of niacin-related compounds

We investigated whether niacin-related compounds had radical-scavenging activity by electron spin resonance methods. Many compounds, but not trigonelline, had radical-scavenging activity against hydroxyl radicals. However, for the nitric oxide radical and 1,1-diphenyl-2-picrylhydrazyl radical, only nicotinic acid hydrazide and isonicotinic acid hydrazide had scavenging activities. These results suggest that the moiety of hydrazide might have an important role in scavenging abilities of various radicals.