Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs

The effects of several imidazole antimycotic agents, an imidazole and several mineralocorticoid analogs on the cytochrome P-450(11)beta-catalyzed 11 beta-hydroxylation of 11-deoxycorticosterone and aldosterone synthesis were examined. Ketoconazole, clotrimazole, miconazole and etomidate were found to be potent inhibitors of the reactions, causing 50% inhibition of the 11 beta-hydroxylase activity at concentrations between 10(-8) and 10(-7) M. The potency of etomidate as to the inhibition of aldosterone- and 18-hydroxycorticosterone-production was found to be almost equal to that in the case of 11 beta-hydroxylation. Spironolactone and other newly synthesized mineralocorticoid analogs were also found to inhibit the cytochrome P-450(11)beta-mediated reactions. The ID50 values of these drugs for inhibition of the 11 beta-hydroxylase activity were almost equal to those in the case of the aldosterone- and 18-hydroxycorticosterone-biosynthetic activities. The results of kinetical studies indicated that one of the mineralocorticoid analogs, Compound 23-0586, acts as a competitive inhibitor for the cytochrome P-450(11)beta-mediated reactions.     

Morphometric analysis of the genusHemerocallis L. (Liliaceae) in Korea

To better understand the patterns of variability and distributions ofHemerocallis in Korea, 53 locations were visited and measurements of 19 morphological and phenological characters were taken on plants directly from their natural habitats. For morphometric analysis, 10 plants from each of 34 populations and five herbarium specimens ofH. middendorffii were used and the data from 12 quantitative characters was analyzed using univariate analysis. Except the littoral populations of Cheju, Hong, Taehuksan, and Sohuksan Islands (H. hongdoensis M. Chung & S. Kang), three peninsular KoreanHemerocallis species can be recognized mainly in South Korea:H. hakuunensis Nakai (=H. micrantha Nakai, growing on southern, central, and northwestern Korea);H. thunbergii Baker (=H. coreana Nakai, found on southeastern and central Korea); andH. middendorffii Tr. et Mey. (central and northeastern Korea). Morphological and phenological features contributing to recognition of the three groups were; color of perianth, shape of roots, shape of inflorescence, flowering time, odor, length of inflorescence, width of the lowest bracts, length of perianth tube enclosing a ovary, width of the inner perianth lobes. Natural hybridization seems to be rare in KoreanHemerocallis. It appears that the KoreanHemerocallis species are relatively well characterized by their distribution patterns, phenology, and habitats compared with the JapaneseHemerocallis species.     

Spectral study of b cytochromes in yeast mitochondria and intact cells

Three types of b cytochromes are demonstrated in Candida utilis mitochondria. One of these b cytochromes has a symmetrical -band at 561.5 nm at room temperature. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate both in coupled and uncoupled mitochondria. The second b cytochrome has a double -band at 565 nm and 558 nm. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate in coupled mitochondria, but in uncoupled mitochondria it is slowly reduced after anaerobiosis and this reduction rate is enhanced by antimycin A addition. Thus the oxidation-reduction state of this cytochrome is energy dependent. The first cytochrome is spectroscopically identified as cytochrome b_K and the second as cytochrome b_T. The third b cytochrome has an -band around 563 nm (b₅₆₃) and is reduced slowly after anaerobiosis in uncoupled mitochondria but faster than the b_T. Further properties of this component are not known. Midpoint potentials of cytochromes b_T, b₅₆₃ and b_K are approximately −50 mV, +5 mV, and +65 mV, respectively.

In intact cells, cytochrome b_T is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome b_T while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome b_T.     

Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis

The vacuole occupies most of the volume of plant cells; thus, the tonoplast marker delta-tonoplast intrinsic protein-green fluorescent protein delineates cell shape, for example, in epidermis. This permits rapid identification of mutants. Using this strategy, we identified the cell shape phenotype-1 (csp-1) mutant in Arabidopsis thaliana. Beyond an absence of lobes in pavement cells, phenotypes included reduced trichome branching, altered leaf serration and stem branching, and increased stomatal density. This result from a point mutation in AtTPS6 encoding a conserved amino-terminal domain, thought to catalyze trehalose-6-phosphate synthesis and a carboxy-terminal phosphatase domain, is catalyzing a two-step conversion to trehalose. Expression of AtTPS6 in the Saccharomyces cerevisiae mutants tps1 (encoding a synthase domain) and tps2 (encoding synthase and phosphatase domains) indicates that AtTPS6 is an active trehalose synthase. AtTPS6 fully complemented defects in csp-1. Mutations in class I genes (AtTPS1-AtTPS4) indicate a role in regulating starch storage, resistance to drought, and inflorescence architecture. Class II genes (AtTPS5-AtTPS11) encode multifunctional enzymes having synthase and phosphatase activity. We show that class II AtTPS6 regulates plant architecture, shape of epidermal pavement cells, and branching of trichomes. Thus, beyond a role in development, we demonstrate that the class II gene AtTPS6 is important for controlling cellular morphogenesis.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

SEC14 Phospholipid Transfer Protein Is Involved in Lipid Signaling-Mediated Plant Immune Responses in Nicotiana benthamiana

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.     

Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan.

Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.