The biological activity and tissue distribution of 2',3'-dihydrophylloquinone in rats

2',3'-Dihydrophylloquinone (dihydro-K1) is a hydrogenated form of vitamin K1 (K1), which is produced during the hydrogenation of K1-rich plant oils. In this study, we found that dihydro-K1 counteracts the sodium warfarin-induced prolonged blood coagulation in rats. This indicates that dihydro-K1 functions as a cofactor in the posttranslational gamma-carboxylation of the vitamin K-dependent coagulation factors. It was also found that dihydro-K1 as well as K1 inhibits the decreasing effects of warfarin on the serum total osteocalcin level. In rats, dihydro-K1 is well absorbed and detected in the tissues of the brain, pancreas, kidney, testis, abdominal aorta, liver and femur. K1 is converted to menaquinone-4 (MK-4) in all the above-mentioned tissues, but dihydro-K1 is not. The unique characteristic of dihydro-K1 possessing vitamin K activity and not being converted to MK-4 would be useful in revealing the as yet undetermined physiological function of the conversion of K1 to MK-4.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Maximization of hydrogen production ability in high-density suspension of Rhodovulum sulfidophilum cells using intracellular poly(3-hydroxybutyrate) as sole substrate

Growth of and hydrogen production by wild-type (WT) Rhodovulum sulfidophilum were compared with those by one of its mutants lacking the poly(3-hydroxybutyrate) (PHB) biosynthesis ability (PNM2). During phototrophic growth under aerobic conditions with fixed illumination, changes in the extinction coefficient and PHB content of WT and PNM2 cells revealed interference of light penetration by PHB. WT cells synthesized PHB at an early stage of the cultivation. PHB degradation after exhaustion of acetate during the cultivation of WT resulted in a decrease of the extinction coefficient. The hydrogen production rate under anaerobic conditions with fixed illumination was examined in WT and PNM2 cell suspensions at different densities. The hydrogen production rate was determined not by the light penetration but by the kinds of hydrogen donors and the density of suspension. The highest value of the rate of hydrogen production from PHB, 33.0 ml/l/h, was improved compared with 26.6 ml/l/h, which was the highest value in hydrogen production from succinate. Under the same illumination, conversion to hydrogen from PHB is more efficient than that from succinate, which is one of the best substrates for hydrogen production. These results suggest that the hydrogen production rate can be maximized in the hydrogen production system based on PHB degradation, which is achieved in high-density suspension under external-substrate-depleted conditions after aerobic cultivation in the presence of an excess amount of acetate.     

Reaction kinetics and modeling of the enzyme-catalyzed production of lactosucrose using beta-fructofuranosidase from Arthrobacter sp. K-1

Lactosucrose synthesis from sucrose and lactose was carried out by using beta-fructofuranosidase from Arthrobacter sp. K-1. The transfructosylation mechanism was found to be of an ordered bi-bi type in which sucrose was bound first to the enzyme and lactosucrose was released last. Hydrolysis side-reaction experiments indicated that the reactions were uncompetitively inhibited by glucose and lactose, while no inhibition by fructose was apparent. The overall reaction rates were formulated. The reaction rate constants, equilibrium constant, and dissociation and Michaelis constants were determined at 35 degrees C and 50 degrees C by fitting the experimental concentration changes with the calculated values by a nonlinear least-square method. The average relative derivation for the concentrations was 9.67%. The kinetic parameters were also calculated for 43 degrees C and 60 degrees C by assuming the Arrhenius law, and the course of reaction was predicted. The obtained reaction rate equations well represented the concentration changes during the experiment at all temperatures.     

Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and the increase of their expression by heat shock and exposure to a hazardous chemical

We isolated and analysed two genomic DNAs that encode the heat-shock protein Hsp30 from Coriolus versicolor. The amino acid sequences substitute only three amino acid substitutions. The promoter regions contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. The levels of mRNAs for Hsp30 increased markedly after exposure of C. versicolor to pentachlorophenol and levels were higher than those after heat shock.     

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.     

Establishment and characterization of a cell-line originated from human mucinous cystadenocarcinoma of the ovary

We recently established a cell line (designated 371M) derived from an ovarian mucinous cystadenocarcinoma. The tumor cells were obtained from the ascitic fluid of a 54-year-old Japanese woman while she was undergoing surgery. Adjuvant chemotherapy (combined paclitaxel and carboplatin) was administered, but was ineffective, and she died about 4 months after surgery. The 371M cells continuously propagated in vitro over a period of about 50 months and, to date, have undergone over 100 passages. They proliferated in a monolayered sheet with doubling times of 84 h and 37 h in the 10th and 34th passages, respectively. When transplanted into nude mice, the tumor histopathologically resembled the structure of the original tumor. The 371M cells secreted high levels of CA125 and CA19-9 into the culture medium. There were several abnormal chromosomes in all karyotypes selected at random. Sensitivity of 371M cells to a variety of anti-cancer drugs was examined by in vitro MTT assay, and the results suggested that CPT-11 and CDDP were more effective against 371M cells than other anti-cancer agents.     

Beraprost sodium,a prostaglandin I2 analogue,protects against advanced gycation end products-induced injury in cultured retinal pericytes

BACKGROUND: Beraprost sodium, a prostaglandin I2 analogue, has been recently reported to exhibit beneficial effects on atherosclerosis in patients with diabetes. However, effects of beraprost sodium on microvascular injury in diabetes remain to be elucidated. We have previously shown that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, caused pericyte apoptosis, thus being involved in the pathogenesis of the early phase of diabetic retinopathy. In this study, we examined whether beraprost sodium can protect against AGE-induced cytotoxicity in cultured retinal pericytes. MATERIALS AND METHODS: Intracellular formation of reactive oxygen species (ROS) was detected using a fluorescent probe. DNA synthesis was determined by measuring [3H]thymidine incorporation into cells. Apoptosis was determined by DNA fragmentations, which were quantitatively measured in an enzyme-linked immunosorbent assay. RESULTS: Beraprost sodium or forskolin, a stimulator of adenylate cyclase, was found to significantly inhibit AGE-induced ROS generation and the subsequent decrease in DNA synthesis in pericytes. Both treatments significantly prevented AGE-induced apoptotic cell death in pericytes. Furthermore, beraprost sodium was found to down-regulate AGE receptor mRNA levels in pericytes. CONCLUSION: The results demonstrated that cyclic AMP-elevating agents such as beraprost sodium and forskolin protected retinal pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that beraprost sodium may have therapeutic potentials in treatment of patients with early diabetic retinopathy.     

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase

Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known. Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV. The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. Based on the model structure, we determined the following: (1) the enzyme requires a Zn(2+) ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.