cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase,a critical enzyme of the legume isoflavonoid phytoalexin pathway

Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4'-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.     

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.     

Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants

The decrease in catalase activity and its relationship to change in salicylic acid content were investigated in rice, wheat, and cucumber seedlings exposed to oxidative stresses. A decrease in chlorophyll fluorescence (F/Fm), measured as an indicator of the oxidative stress, and a drop in catalase activity were observed following treatment with NaCl in all plant seedlings tested . Furthermore, such decreases in F/Fm and catalase activity were also observed under low temperature conditions in both rice cultivars, whereas the degrees of decrease were dependent on their low temperature tolerance . Although the content of salicylic acid increased in rice seedlings stressed by NaCl treatment, it was inversely correlated with the decrease in the catalase activity . Such a relationship between the decrease in catalase activity and increase in salicylic acid content was confirmed with paraquat treatment of the rice seedlings . These results suggested that the fall in catalase activity is a phenomenon occurring in many plant species under oxidative stress and is related to the accumulation of salicylic acid in oxidatively-stressed plants.     

Direct analysis of drugs in plasma by column-switching liquid chromatography-mass spectrometry using a methylcellulose-immobilized reversed-phase pretreatment column

A novel methylcellulose-immobilized reversed-phase pretreatment column (MC-ODS) for column switching liquid chromatography-mass spectrometry (LC-MS) was investigated to improve recovery and durability. Pretreatment and analytical conditions were optimized so that high throughput and high selectivity was ensured during mass spectrometric analysis. Analytical runs, including deproteinization and gradient LC analysis, were conducted in a 6-min cycle. As a consequence, recoveries for test drugs (metoprolol, propranolol, lidocaine, dibucaine, bupivacaine) were greater than 90% and more than 300 plasma samples spiked with target compounds were directly injected and measured without compromising MS detection or system performance.     

Pigmentation pattern formation in butterflies: experiments and models

Butterfly pigmentation patterns are one of the most spectacular and vivid examples of pattern formation in biology. They have attracted much attention from experimentalists and theoreticians, who have tried to understand the underlying genetic, chemical and physical processes that lead to patterning. In this paper, we present a brief review of this field by first considering the generation of the localised, eyespot, patterns and then the formation of more globally controlled patterns. We present some new results applied to pattern formation on the wing of the mimetic butterfly Papilio dardanus.     

Antimicrobial activity of essential oils against Helicobacter pylori

Background. Helicobacter pylori is an important pathogen responsible for gastroduodenal diseases in humans. Although the eradication of H. pylori using antibiotics often improves gastroduodenal diseases, resistance to the antibiotics is emerging. Materials and Methods. The antimicrobial effect of essential oils and the development of resistance to the essential oils were evaluated in vitro and in vivo. Results. Thirteen essential oils used in this study completely inhibited the growth of H. pylori in vitro at a concentration of 0.1% (v/v). Cymbopogon citratus (lemongrass) and Lippia citriodora (lemon verbena) were bactericidal against H. pylori at 0.01% at pH 4.0 and 5.0. Resistance to lemongrass did not develop even after 10 sequential passages, whereas resistance to clarithromycin developed under the same conditions. In in vivo studies, the density of H. pylori in the stomach of mice treated with lemongrass was significantly reduced compared with untreated mice. Conclusions. These results demonstrate that the essential oils are bactericidal against H. pylori without the development of acquired resistance, suggesting that essential oils may have potential as new and safe agents for inclusion in anti‐H. pylori regimens.     

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.     

Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.