Presence of carnitine acetyltransferase in peroxisomes and in mitochondria of oleic acid-grown Saccharomyces cerevisiae

Abstract Paracoccidiodes brasiliensis , the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N -acetlyneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin ( Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 × 10⁶ residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.     

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry.The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.     

Ursodeoxycholic acid protects hepatocytes against oxidative injury via induction of antioxidants.

The therapeutic efficacy of ursodeoxycholic acid (UDCA) has been widely demonstrated in various liver diseases, suggesting that UDCA might protect hepatocytes against common mechanisms of liver damage. A candidate for such protection is oxidative injury induced by reactive oxygen species. This study was designed to assess the effects of UDCA on oxidative injury and antioxidative systems in cultured rat hepatocytes. The viability of the hepatocytes dose-dependently decreased after hydrogen peroxide or cadmium administration. Pretreatment with UDCA significantly prevented this decrease in viability. The amounts of glutathione (GSH) and protein thiol increased significantly, but the activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and catalase were unchanged in UDCA-treated hepatocytes. The mRNA levels of gamma-glutamylcysteine synthetase and metallothionein (MT) were significantly higher in UDCA-treated hepatocytes than in controls. In conclusion, UDCA increased hepatocyte levels of GSH and thiol-containing proteins such as MT, thereby protecting hepatocytes against oxidative injury. Our results provide a new perspective on the hepatoprotective effect of UDCA.     

Production of autoproteolytically subunit-assembled 7-β-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. C427 using a chitin-binding domain

7--(4-Carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 is known as a proteolytically processed bacterial enzyme. GL-7ACA acylase from Pseudomonas sp. C427 (C427) consists of - and -subunits that are processed from a precursor peptide by removing the spacer peptide. A chitin-binding domain (CBD) of chitinase A1 derived from Bacillus circulans was genetically fused into four different positions of the C427-encoding gene. In the four enzymes thereby produced, N427, SP427, C427, and C427, it was fused, respectively, to the N-terminal region of the -subunit; the C-terminal region of the -subunit; the three-amino-acid upper region of the C-terminal of the -subunit; and to the C-terminal region of the -subunit. All of the fusion enzymes, expressed in Eschericha coli, were successfully processed into active forms and had GL-7ACA acylase activity. The affinity-binding activity to crystalline chitin was affected by the fusing position of CBD. N427, C427, and C427 remained fused to the CBD after their processing steps and could bind to chitin, but in the case of SP427 the fused CBD was cleaved away during the processing steps and binding activity was no longer observed. These results indicate that CBD is functional in such autoproteolytically subunit-assembled acylases.     

Life-history traits of the acarophagous lady beetle,<Emphasis Type="Italic">Stethorus japonicus</Emphasis> at three constant temperatures

Stethorus japonicusKamiya (Coleoptera: Coccinellidae) is an indigenous ladybird beetle in Japan, which feeds on many spider mite species. We evaluated the development, survivorship and life-history parameters of this lady beetle on a diet of eggs of the two-spotted spider mite, Tetranychus urticae Koch (red form) (Acari: Tetranychidae). In addition, the effect of short photoperiod on its reproduction was assessed. Survival rates from egg to adult were more than 71% at temperatures between 17.5 and 30 °C. The highest immature mortality was 100% at 35 °C followed by 76% at 15 °C and 52% at 32.5 °C. The lower threshold temperature for development from egg to egg-laying adult was 13.0 °C and the thermal constant was calculated as 238.7° days. Based on these data, the maximum number of generations that could complete development in a year under field conditions in Ibaraki, central Japan, would be between five and seven. The intrinsic rates of natural increase (r_m) were 0.093 at 20 °C, 0.156 at 25 °C and 0.241 at 30 °C. Reproductive diapause was induced at photoperiods with light phases shorter than 13 h at 18 °C.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from<Emphasis Type="Italic"> Bacillus</Emphasis> spp.

Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.Communicated by K. Horikoshi     

Genetic evaluation of peroxisomal and cytosolic acetoacetyl-CoA thiolase isozymes in n-alkane-assimilating diploid yeast, Candida tropicalis

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic. Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrated that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.     

Enhancement of carnitine acetyltransferase synthesis in alkane-grown cells and propionate-grown cells of Candida tropicalis

The level of carnitine acetyltransferase was markedly increased in harmony with appearance of peroxisomes in alkane-grown cells and propionate-grown cells of Candida tropicalis. From immunochemical studies with antibodies against peroxisomal and mitochondrial carnitine acetyltransferases, it was confirmed that no other type of the enzyme than the peroxisomal and mitochondrial ones was present in alkane-, propionate- and glucose-grown cells of the yeast. The increase in the enzyme level in alkane- and propionate-grown cells was immunochemically proved to result from the increase in the amount of the enzyme protein.Dedicated to Professor Hans G. Schlegel on occasion of his 60th birthday