Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.     

Impaired development of mammary glands in scorbutic rats unable to synthesize ascorbic acid

The effects of ascorbic acid (AsA)-deficiency on the development of mammary glands were investigated using mutant rats (osteogenic disorder syndrome rats; ODS rats) with hereditary inability to synthesize AsA. Female ODS rats of 21 days old were castrated and divided into two groups. One group was given AsA in their drinking water, and the other was not. All the rats received a daily injection of oestradiol-17 beta and progesterone (EP) from day 28 to day 49 of age. After EP treatment, the concentrations of AsA in the mammary glands of rats not given AsA were less than one tenth of those of rats given AsA and the contents of hydroxyproline in the mammary glands of the former rats were about half of those in the latter. Furthermore, the concentration of serum prolactin in rats not given AsA was reduced to about one third of that in rats given AsA. After EP treatment, whole mounts of mammary glands showed that in rats not given AsA the development of ducts was impaired and there was extensive accumulation of endbuds. Consistent with this finding, EP injections did not increase the area of parenchyma in the mammary glands of rats not given AsA, whereas they increased it about 2-fold in rats given AsA. Moreover, after EP treatment the amount of alpha-lactalbumin was significantly less in the mammary parenchyma of rats not given AsA than in that of rats given AsA. On the other hand, AsA deficiency did not impair the response of the mammary cells to insulin or prolactin in terms of DNA synthesis and alpha-lactalbumin production. These findings indicate that AsA deficiency impaired the development of mammary glands. This effect may be partly attributable to a defect in collagen synthesis in the mammary glands and a decrease in the concentration of serum prolactin.     

Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01–1.0μg/ml of leukotriene C4 and 12.5–400μg/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0μg/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6, 7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole     

Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA.

Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4. The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha. It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs. There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA. It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA.     

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria

Ascorbate (AsA) peroxidase was found in six species of cyanobacteriaamong ten species tested. Upon the addition of H₂¹⁸O₂ to thecells of AsA peroxidase-containing cyanobacteria, ¹⁶O₂ derivedfrom water and ¹⁸O₂ derived from H₂^I8O₂ were evolved in thelight. The evolution of ¹⁶O₂ was inhibited by DCMU and did notoccur in the dark, but ^I8O₂ was evolved even in the dark orin the presence of DCMU. Similar light-dependent evolution of¹⁶O₂ was observed in the cells of AsA peroxidase-containingEuglena and Chlamydomonas. However, the cells of AsA perox-idase-lackingcyanobacteria evolved only ¹⁸O₂ in either the light or dark.Furthermore, the quenching of chlorophyll fluorescence inducedby hydrogen peroxide was observed only in the cells of the AsAperoxidase-containing Synechocystis 6803, and not in the cellsof Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteriacan be divided into two groups, those that has and those thatlacks AsA peroxidase. The first group scavenges hydrogen peroxidewith the peroxidase using a photoreductant as the electron donor,and the second group only scavenges hydrogen peroxide with catalase. (Received July 23, 1990; Accepted October 18, 1990)     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

Cell-surface hydrophobicity of Candida species as determined by the contact-angle and hydrocarbon-adherence methods

Cell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.