A zinc-binding citrus protein metallothionein can act as a plant defense factor by controlling host-selective ACR-toxin production

Metallothionein is a small cysteine-rich protein known to have a metal-binding function. We isolated three different lengths of rough lemon cDNAs encoding a metallothionein (RlemMT1, RlemMT2 and RlemMT3), and only RlemMT1-recombinant protein had zinc-binding activity. Appropriate concentration of zinc is an essential micronutrient for living organisms, while excess zinc is toxic. Zinc also stimulates the production of host-selective ACR-toxin for citrus leaf spot pathogen of Alternaria alternata rough lemon pathotype. Trapping of zinc by RlemMT1-recombinant protein or by a zinc-scavenging agent in the culture medium caused suppression of ACR-toxin production by the fungus. Since ACR-toxin is the disease determinant for A. alternata rough lemon pathotype, addition of RlemMT1 to the inoculum suspension led to a significant decrease in symptoms on rough lemon leaves as a result of reduced ACR-toxin production from the zinc trap around infection sites. RlemMT1-overexpression mutant of A. alternata rough lemon pathotype also produced less ACR-toxin and reduced virulence on rough lemon. This suppression was caused by an interruption of zinc absorption by cells from the trapping of the mineral by RlemMT1 and an excess supplement of ZnSO₄ restored toxin production and pathogenicity. Based on these results, we propose that zinc adsorbents including metallothionein likely can act as a plant defense factor by controlling toxin biosynthesis via inhibition of zinc absorption by the pathogen.     

Dosing Time‐Dependent Effect of Raloxifene on Plasma Fibrinogen Concentration in Ovariectomized Rats

The chronopharmacological effect of raloxifene, a selective estrogen‐receptor modulator, was evaluated by repeated dosing of ovariectomized rats. Bilateral ovariectomy or sham operation was performed at age 12 wks, and animals were kept in rooms with a 12 h light‐12 h dark cycle. Raloxifene (3 mg/kg, once daily for 10 wks) or vehicle was given repeatedly at either 2 h after lights‐on (2 HALO) or 14 h after lights‐on (14 HALO). Plasma fibrinogen concentration at the end of the study was reduced by the drug, and the reduction was significantly prominent in rats in whom the drug was dosed at 2 HALO rather than 14 HALO. Femur bone density decreased, and urinary excretion of deoxypyridinoline, an index of bone resorption capacity of osteoclasts, increased in ovariectomized animals at the end of the study. Treatment with raloxifene ameliorated these changes in a dosing time‐independent manner. Serum calcium, ALT, and total protein concentrations at the end of the study also did not differ acccording to treatment regime, which indicates that protein synthesis and liver function may not contribute to the effects. This is the first study to determine dosing time‐dependent changes in the efficacy of raloxifene in an animal model of osteoporosis. Because fibrinogen concentration is reported to be a marker of cardiovascular events, consideration of dosing time of raloxifene may be important to obtain a better cardioprotective effect of this medication when it is prescribed to postmenopausal women with osteoporosis.     

The substrate binding cavity of particulate methane monooxygenase from Methylosinus trichosporium OB3b expresses high enantioselectivity for n-butane and n-pentane oxidation to 2-alcohol

The particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b oxidized n-butane and n-pentane and mainly produced (R)-2-butanol and (R)-2-pentanol that comprised 78 and 89% of the product, respectively, indicating that the pro-R hydrogen of the 2-position carbon of n-butane and n-pentane is oriented toward a catalytic site within the substrate binding site of pMMO. The protein cavity adjacent to the catalytic center for pMMO has optimum volume for recognizing n-butane and n-pentane for enantioselective hydroxylation.     

A monoamine in the gustatory cell of the frog's taste organ

Summary Fluorescence histochemistry reveals that in the frog's taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 m. Following reserpine treatment the yellow fluorescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin).The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700–1000 Å in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions or nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustatory cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.These results were reported in a preliminary form to the October, 1974 meeting of the Japan Society of Histochemistry and Cytochemistry.The authors gratefully acknowledge the support and helpful advice of Prof. Dr. T. Kanaseki.     

High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5′-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3′-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.     

Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D

Antisense suppression is a powerful tool to analyze gene function. In this study, we show that antisense RNA suppressed the expression of a target gene in the unicellular red alga, Cyanidioschyzon merolae. In this study, the antisense strand of the catalase gene was cloned and inserted into an expression vector upstream of the GFP gene. This plasmid was introduced into C. merolae cells using a polyethylene glycol-mediated transformation protocol. Using the expression of GFP as a marker of transformed cells, the expression of catalase was examined by immunocytochemistry. Decreased expression of catalase was observed in cells that were transformed with the antisense strand of the catalase gene. These results indicate the utility of this antisense suppression system.     

Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402

Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 μmol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system.