Ester Formation by Alcohol Acetyltransferase from Brewers’ Yeast

Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers’ yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7 ? 8. It was least active against C₃ alcohol among C₁ ? C₆ alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.     

Lipid Phase Fatty Acid Flip-Flop, Is It Fast Enough for Cellular Transport?

The mechanism by which fatty acids are transported across cell membranes is controversial. The essence of the controversy is whether transport requires membrane protein mediation or whether the membrane's lipid phase provides a pathway so rapid that a protein is not needed. This review focuses on the mechanisms of fatty acid transport across lipid bilayer membranes. These results for lipid membranes are used to help evaluate transport across cell membranes. Within the context of this analysis, a lipid phase mediated process is consistent with results for the transport of fatty acids across erythrocytes but provides a less adequate explanation for fatty acid transport across more complex cells. Received: 16 June 1999/Revised: 21 January 2000     

Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.     

Dissociation and Reassociation of Xanthomonas α-Amino Acid Ester Hydrolase

We isolated a cDNA for basic class I chitinase (ChitiWb1). ChitiWb1 cDNA encodes a protein that consists of 315 amino acid residues and has a signal peptide. Northern blot analysis indicated that the class I chitinase mRNA in leaves and cultured cells of winged bean was increased by treatments with NaCl, KCl, CaCl₂, mannitol or saccharose, but not with abscisic acid. Thus, class I chitinase expression was shown to be up-regulated by osmotic stress.     

Trehalose Lipid and α-Branched-β-hydroxy Fatty Acid Formed by Bacteria Grown on n-Alkanes

A bacterium isolated in our laboratory, Arthrobacter paraffineus KY 4303. when grown on n-paraffin as the sole source of carbon, produced anthrone-positive lipid in the emulsion layer (holding bacterial cells, lipids and n-paraffin remained) of the culture medium. This was isolated and identified as α-branched-β-hydroxy fatty acid trehalose ester.

The addition of penicillin to the growing culture caused a significant suppression of trehalose lipid formation and led consequently to the accumulation of both the precursors, α, α-trehalose and α-branched-β-hydroxy fatty acid, in the culture medium.

The formation of trehalose lipid was also observed in other bacteria which can utilize n-paraffin as the sole source of carbon. In addition, a possible role of this trehalose lipid in the utilization of n-paraffin by these bacteria was discussed.     

5′-Phosphodiesterase Formation by Cultured Plant Cells

5′-Phosphodiesterase (5′-PDase) which degrades RNA to nucleoside-5′-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5 mg of 2,4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5′-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.

The 5′-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5′-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5′-PDase activity as the mother plant on dry cell weight basis.     

Cellular DNA damage by nitrosocimetidine: A comparison with N-methyl-N′-nitroso-nitrosoguanidine and X-irradiation

Permanently proliferating lymphoblastoid cell lines (LCLs) and normal unstimulated peripheral blood leukocytes have been used to study the effects of nitrosocimetidine (NC) on cultured human lymphoid cells. The approaches that were used to assess the cells' ability to cope with NC were: (i) determination of cell survival as measured by colony formation in microtiter plates; (ii) quantitation of DNA synthesis and DNA-repair replication by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (iii) measurement of the induction of alkali labile lesions and strand breaks by NC in ³H-labeled DNA using velocity sedimentation in alkaline sucrose. In summary, treatment with NC was found to inhibit both replicative DNA synthesis and colony formation in LCLs. At the molecular level, NC treatment induced alkali labile lesions in LCL DNA and elicited DNA-repair replication in proliferating LCLs as well as unstimulated lymphocytes. Considered in total, these data indicate that NC is reactive with human DNA in the cellular environment in a manner similar to methylating nitroso compounds which have been shown to be carcinogenic. The significance of these findings will be discussed.     

Degradation of Cellular Phospholipids and Softening of Pressed Baker’s Yeast

Variations in lipid components of washings and homogenate of pressed baker’s yeast were investigated during the storage of pressed baker’s yeast at 30°C. Washings represents the substances which had leaked out from cells. Homogenate represents those contained in whole cells. Lipids in yeast washings increased toward softening, the phospholipids in yeast homogenate decreased continuously during storage. Two stages, an earlier period of storage (Stage I) and a later period of storage (Stage II) were observed in the degradation of phospholipids. Free fatty acid which was the main degradation product of phospholipid accumulated in Stage II, particularly at softening. The order in phospholipid degradation was PC>PE>PI + PS (PI>PS). Moreover, when washings of stored yeast at softening were assayed using ¹⁴C-acyl PC, the release of ¹⁴C-acyl fatty acid was observed.

These results suggest that phospholipids were degraded by some phospholipid-deacylating enzymes toward softening. From the results of lipid analysis, we inferred that the responsible enzymes were phospholipases.     

Identification of High γ-Aminobutyric Acid Producing Marine Yeast Strains by Physiological and Biochemical Characteristics and Gene Sequence Analyses

Four marine yeasts isolated from the Pacific Ocean off Japan (Siki No. 4, Siki No. 15, Hach No. 6, and Inub No. 11), which showed high γ-aminobutyric acid (GABA) producing abilities, were identified and classified by physiological and biochemical characteristics and gene sequence analyses. Analysis of biochemical data suggested that while Siki No. 15 was identical to Candida, the remaining three isolates belonged to the genus Pichia. However, these data were insufficient to resolve their identity at the species level. Subsequently, analysis of the 5.8S rRNA genes and the two internal transcribed spacer regions (ITS) sequences revealed that Siki No. 15 belongs to Pichia guilliermondii, while the remaining three isolates corresponded to Pichia anomala. Since Siki No. 4 showed slightly different biochemical properties than the other two isolates, which were otherwise identical, we sought to investigate the sequences of the intergenic spacer region 1 (IGS1). We observed few nucleotide changes, suggesting that the Hach No. 6 and Inub No. 11 isolates belong to different but new strains for which we propose the names P. anomola MR-1 and MR-2 respectively.     

Evidence for an Ester Linkage between Lignin and Glucuronic Acid in Lignin–Carbohydrate Complexes by DDQ-Oxidation

The effects of ethanol feeding on the tryptophan-niacin metabolism were investigated in rats. Male rats of the Wistar strain (3 weeks old) were fed with a 20% casein diet and 15% ethanol ad libitum for 56 days. Urine samples were collected every week during the experimental period. Urinary excretion of N¹-methylnicotinamide (MNA) was always higher in the ethanol-fed group than in the control group, but urinary excretion of its oxidized metabolites N¹-methyl-2-pyridone-5-carboxamide (2-Py) and N¹-methyl-4-pyridone-3-carboxamide (4-Py) was always lower. Therefore, the ratio of (2-Py + 4-Py)/MNA excretion was lower in the ethanol-fed group than in the control group. The rats were killed on day 57 and liver enzyme activities involved in the tryptophan-niacin metabolism were also measured. Tryptophan oxygenase, kynureninase, nicotinamide mononucleotide adenylyltransferase, NAD⁺ synthetase, and nicotinamide methyltransferase activities were similar in both groups, but, 2-Py-forming MNA oxidase and 4-Py-forming MNA oxidase activities in the ethanol-fed group were 43% and 18% of the control, respectively. Therefore, the increase in MNA excretion and the decrease in the ratio of (2-Py + 4-Py)/MNA excretion might be attributed to the inhibition of the two MNA oxidase activities by ethanol feeding. Furthermore, it happened to be found that this excretion ratio also increased with growth up to nine weeks and this change was attributed to the increased reaction MNA → 4-Py with growth.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Effects Various Adsorbents on Mycelium Formation and Mycophenolic Acid Production by Pénicillium brevicompactum

A drug-resistant mutant, No. 4–23–11, which had been derived from Pénicillium brevicompactum ATCC 16024, was cultured in a liquid medium for the production of mycophenolic acid (MPA) in the presence and absence of various adsorbents, that is, celites, zeolites, aluminas, talc, silica, charcoals, carbon blacks, natural graphite and carbonaceous mesophase spheres. In the absence of an adsorbent, MPA production (0.2 ~4.8 g/1) and the size of mycelium pellets (5~0.5mm) markedly depended on the concentration of spores inoculated (10⁴~ 10⁷/ml), whereas in the presence of one of these adsorbents, small pellets (smaller than 1 mm in diameter) were formed and the high production of MPA (4.5 — 5.4 g/1) was observed, independently of the spore concentration between approximately 10⁴~ 10⁷/ml. Microscopic observation of the pellets revealed that numerous particles of the adsorbents adhered to the surface of the hyphae. These results suggest that the adsorbents might interfere with the adhesion of hyphae to each other and thus retard the formation of large pellets.     

Effect of Globotriaosyl Ceramide Fatty Acid α-Hydroxylation on the Binding by Verotoxin 1 and Verotoxin 2

Variation in the lipid moiety of the verotoxin (VT) receptor glycosphingolipid, globotriaosyl ceramide (Gb₃) can modulate toxin binding. The binding of VT1 and VT2 to C18 and C22 ahydroxy and nonhydroxy fatty acid isoforms of Gb₃ were compared using a receptor ELISA and a ¹²⁵l-labeled toxin/glycolipid microtitre plate direct binding assay. Increased binding to the hydroxylated species, particularly C22OH, was observed for both toxins. Increased RELISA binding at low glycolipid concentrations only, suggested the binding affinity is increased following Gb₃ fatty acid hydroxylation. Nonlinear regression analysis of direct binding assay to these Gb₃ isoforms confirmed the increased affinity of both toxins for the C22 hydroxylated Gb₃. The capacity was also significantly increased. The increased binding of VTs for hydroxylated fatty acid Gb₃ isoforms may be a factor in the selective renal pathology which can follow systemic verotoxemia, particularly in the mouse model. The more pronounced effect at lower glycolipid concentrations prompted investigation of VT1 binding affinity at different Gb₃ concentrations. Unexpectedly, the VT1 Kd for Gb₃ was found to decrease as an inverse function of the Gb₃ concentration. This shows that glycolipids have nonclassical receptor properties.     

2′-O,4′-C-Ethylene-Bridged Nucleic Acid (ENATM) for Effective Antisense Formation

Abstract

ENA^TM antisense oligonucleotides for vascular endothelial growth factor (VEGF) mRNA were synthesized and evaluated in A549 lung cancer cells. It was found that the VEGF ENA-antisense inhibited not only the expression of VEGF, but also the expression of three genes, which were found in Genbank by BLAST and Clustal W search and considered likely to bind to the VEGF ENA-antisense. These results indicate that ENA-antisense oligonucleotides act in a sequence-specific manner, and could be used as effective antisense drugs.     

Formation of Itraconazole–Succinic Acid Cocrystals by Gas Antisolvent Cocrystallization

Cocrystals of itraconazole, an antifungal drug with poor bioavailability, and succinic acid, a water-soluble dicarboxylic acid, were formed by gas antisolvent (GAS) cocrystallization using pressurized CO₂ to improve itraconazole dissolution. In this study, itraconazole and succinic acid were simultaneously dissolved in a liquid solvent, tetrahydrofuran, at ambient conditions. The solution was then pressurized with CO₂, which decreased the solvating power of tetrahydrofuran and caused crystallization of itraconazole–succinic acid cocrystals. The cocrystals prepared by GAS cocrystallization were compared to those produced using a traditional liquid antisolvent, n-heptane, for crystallinity, chemical structure, thermal behavior, size and surface morphology, potential clinical relevance, and stability. Powder X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy analyses showed that itraconazole–succinic acid cocrystals with physical and chemical properties similar to cocrystals produced using a traditional liquid antisolvent technique can be prepared by CO₂ antisolvent cocrystallization. The dissolution profile of itraconazole was significantly enhanced through GAS cocrystallization with succinic acid, achieving over 90% dissolution in less than 2 h. The cocrystals appeared stable against thermal stress for up to 4 weeks under accelerated stability conditions, showing only moderate decreases in their degree of crystallinity but no change in their crystalline structure. This study shows the utility of an itraconazole–succinic acid cocrystal for improving itraconazole bioavailability while also demonstrating the potential for CO₂ to replace traditional liquid antisolvents in cocrystal preparation, thus making cocrystal production more environmentally benign and scale-up more feasible.KEY WORDS: cocrystals, dissolution rate, gas antisolvent, itraconazole     

A Fox2-Dependent Fatty Acid ?-Oxidation Pathway Coexists Both in Peroxisomes and Mitochondria of the Ascomycete Yeast Candida lusitaniae

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying ¹⁴C_α-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.     

Acylation of Bowman–Birk Soybean Proteinase Inhibitor by Unsaturated Fatty Acid Derivatives

A procedure was developed for acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by N-hydroxysuc-cinimide esters of oleic, linoleic, and -linolenic acids in a dimethyl sulfoxide–dioxane–pyridine mixture. BBI derivatives containing two acylated amino groups were prepared with high yield. The use of the reversible modifier citraconic anhydride in the first stage of synthesis permitted the synthesis of hydrophobized BBI derivatives retaining high antitrypsin and antichymotrypsin activities. It was found that the insertion of two long chain moieties in the BBI molecule decreases its thermostability.