Molecular and Biochemical Characterization of Three Anthocyanin Synthetic Enzymes from Gentiana triflora

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol4-reductase and flavonoid 3-glucosyltransferase were clonedfrom petals of Gentiana triflora. Their sequences were homologousto counterparts from other plants. Flavonoid 3',5'-hydroxylaseand flavonoid 3-glucosyltransferase were enzymatically characterizedby expressing cDNAs in heterologous expression systems. (Received May 21, 1996; Accepted June 4, 1996)     

Synthesis and evaluation of bioactive naphthoquinones from the Brazilian medicinal plant, Tabebuia avellanedae

A series of naphthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (−)-5-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1) and its positional isomer, (−)-8-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (2), which are secondary metabolites found in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited potent antiproliferative effect against several human tumor cell lines, but its effect against some human normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5 is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer chemopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicrobial activity. Both compounds displayed modest antifungal and antibacterial activity (Gram-positive bacteria), whereas they were inactive against Gram-negative bacteria.     

cDNA of a Novel mRNA Expressed Predominantly in Mouse Kidney

We examined embryonic carcinoma (EC) cells for a potential prototype molecule of C3, the third component of complement. PCR primers, corresponding to the base sequence derived from the C3 cDNA of several species, were used for PCR amplification of the EC cell cDNA. All the PCR products obtained had the same sequence and showed no sequence homology to C3. Subsequently, cDNA clones were isolated from a mouse liver cDNA library using the PCR product as a probe. Unexpectedly, neither the base sequence of the cDNA clones nor the amino acid sequence deduced from the cDNA showed homology to C3, although partial homology was observed to a number of sequences from EST databases. We designated this new clone NCU-G1. Northern hybridization experiments revealed that NCU-G1 is expressed constitutively not only in the mouse fetus but also in various mouse tissues, and is most abundant in the kidney cortex.     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

Induction and Secretion of Pathogenesis-Related Proteins by Salicylate or Plant Hormones in Tobacco Suspension Cultures

Pathogenesis-related proteins (PR proteins), that are inducedin tobacco leaves in hypersensitive response to infection withtobacco mosaic virus (TMV) or by treatment with chemicals, werefound to be also inducible in a dedifferentiated system, tobaccosuspension culture. Quantitative determination of these proteinsusing anti PR 1a IgG showed that their increase started at aboutthe end of cell growth period and that their production couldbe enhanced by the addition of potassium salicylate, Eosin Yellowishand plant hormones (GA3, IAA and 2,4-D). The production dependedon the concentration of the chemical inducer and the cell lineused. In BY-2 cell line, PR proteins amounted to 12 µgat day 5 and then increased exponentially with time, reaching280 µg or 70 µg per g fr wt of cells at day 9 withor without the addition of 25 µM potassium salicylate.More than 90% of the induced PR proteins was found in the mediumand less than 10% in the cells at day 9. Peroxidase activityin the medium was constant throughout the experiment althoughtotal activity in the flask increased with cell growth, indicatingthat PR proteins are actively secreted into the medium. (Received November 12, 1986; Accepted March 6, 1987)     

An alkaline thiol proteinase in the liver mitochondria of bullfrog, Rana catesbeiana

The mitoplasts were prepared from bullfrog (Rana catesbeiana) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca2+-dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p-chloromercuribenzoate. Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly (ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.