A highly sensitive method for measurement of urinary hyaluronan with a minimum molecular mass of 2,000 Da was developed without using HPLC or radioisotopes. This competitive enzyme-linked immunosorbent assay-like method, used competitive binding of free hyaluronan in the sample and biotin-labeled standard hyaluronan to hyaluronan binding protein in solid phase. A total of 150 healthy individuals from both sexes at ages from 0 to 100 years was examined by the established method. Hyaluronan of 384±80 ng/mg creatinine (mean±SD) was constantly excreted into urine of 24-40-year-old healthy adults. The urinary hyaluronan levels were significantly higher before age 1 (p<0.001) and rather high after 90 years compared to the other groups. The average molecular weight of urinary hyaluronan (5,500 Da) was constant through all generations. Sex difference of urinary hyaluronan was not observed both quantitatively or qualitatively.
Coumaran (2,3-dihydrobenzofuran), a secondary metabolite of Cyperus nipponicus, inhibits the feeding of polyphagous insects. This secondary metabolite is regarded as one of the defensive systems of the Cyperaceae. A number of naturally occurring benzofurans that differ in their substitution pattern and oxidation state have been investigated for their ability to inhibit insect feeding by a bioassay with the common cutworm (Spodoptera litura F. Noctuidae) that applies the leaf disk method. The evaluation of the antifeedant activity of each test compound used the ED50 value based on the dose-response curve that was calculated with the probit method. The 2,3-dihydrobenzofuran derivative, 7-acetyl-4,6-dimethoxy-2-isopropenyl-2,3-dihydrobenzofuran, had an ED50 value of 1.3 μg (5.4×10-9 mol)/cm2 against the common cutworm. The introduction of methoxy and acetyl groups increased the insect antifeedant activity. Furthermore, the insect antifeedant activity increased with decreasing lipophilicity of the test compounds.
Four novel taxane diterpenes were isolated from the needles of the Japanese yew, Taxus cuspidata. Their structures were established as 1-hydroxy taxuspine C, 2α,7β,9α, 10β,13α-pentaacetoxy-5α-cinnamoyloxy-4β,20-epoxy- taxa-11-en-1β-ol, 2α,9α,10β,13α-tetraacetoxy-taxa-4(20), 11-dien-5α-ol and 10-deacetyl taxinine on the basis of spectral analyses.
Our search for new metabolites biosynthetically related to fusicoccin with potent plant growth-stimulating activity from the culture filtrate of Fusicoccum amygdali F6 resulted in the isolation of a new derivative of fusicoccin J, as well as fusicoccin J and 16-O-demethyl 3-epifusicoccin J. Its structure was analyzed by 600 MHz NMR spectrometry and identified as 16-O-demethyl fusicoccin J. The biosynthetic relationships among 16-O-demethyl fusicoccin J and its 3-epimer, and fusicoccin J are discussed in connection with the co-occurrence of both of the 3-epimers of 16-O-demethyl fusicoccin J. 16-O-Demethyl fusicoccin J and its 3-epimer showed only low germination-stimulating activity toward lettuce seeds in the presence of abscisic acid (20 μM), the latter being slightly more active than the former.
5-(3′-Hydroxyphenyl)pentanoic acid (1) and 5-(3′-methoxyphenyl)pentanoic acid (2) occurring in the roots of Athyrium yokoscense showed transport activity to alkaline and alkaline earth metal ions and heavy divalent metal ions.
(+)-(2S,3R)-Piscidic acid was efficiently synthesized with high optical purity (90% e.e.) via Sharpless catalytic asymmetric dihydroxylation of a trisubstituted olefin in only 6 steps from commercially available 4-hydroxyphenylpyruvic acid as the starting material. The reaction proceeded with high optical purity by using the chiral ligands, dihydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl diether or dihydroquinidine 1,4-anthraquinonediyl diether.
D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter. This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium. Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine. The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0. Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band. The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine. The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa. D-Glucosaminitol dehydrogenase required NAD+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates. In 50 mM Tris-HCl buffer (pH 9.0) at 25°C, the Km for D-glucosaminitol, ethanol, and NAD+ were 2.2, 2.0, and 0.08 mM, respectively. The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol. The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sul- fhydryl groups and with Zn2+ ion. The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol.
Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC gene, which is located at a different locus from that of the glr gene in the B. subtilis genome, share a high sequence similarity. The yrpC gene complemented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene. Glutamate racemase activity was found in the extracts of E. coli WM335 clone cells harboring a plasmid, pYRPC1, carrying its gene. Thus, the yrpC gene encodes an isozyme of glutamate racemase of B. subtilis IFO 3336. YrpC is mostly found in an inactive inclusion body in E. coli JM109/pYRPC1 cells. YrpC was solubilized readily, but glutamate racemase activity was only slightly restored. We purified YrpC from the extracts of E. coli JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it. YrpC is a monomeric protein and contains no cofactors, like Glr. Enzymological properties of YrpC, such as the substrate specificity and optimum pH, are also similar to those of Glr. The thermostability of YrpC, however, is considerably lower than that of Glr. In addition, YrpC showed higher affinity and lower catalytic efficiency for L-glutamate than Glr. This is the first example showing the occurrence and properties of a glutamate racemase isozyme.
Cholestyramine is the most widely used bile acid sequestrant in the treatment of hypercholesterolemia. However, cholestyramine has unpleasant side effects as a consequence of its hydrophobic backbone. Therefore, high-capacity bile acid sequestering biopolymers with cationic chitosan derivatives were developed, because electrostatic interactions are important for binding with bile acid anions. Dialkylaminoalkylation and reductive amination of chitosan were done to add dialkylaminoalkyl and an additional free amino group at a hydroxyl site in the chitosan backbone respectively and the aminoderivatized chitosan derivatives were quaternized with methyl iodide to produce a cationic polyelectrolyte. The in vitro bile acid binding capacity of the chitosan derivatives in aqueous NaCl was measured by reversed-phase HPLC. The binding capacities of sodium glycocholate (a major bile acid) to chitosan, DEAE-chitosan, quaternized DEAE-chitosan, and cholestyramine were 1.42, 3.12, 4.06, and 2.78 mmol/g resin, respectively. With quaternized DEAE-chitosan, the bile acid binding capacity increased ~50% over that of cholestyramine. The bile acid binding capacity of dialkylaminoalkyl chitosan derivatives increased with the number of carbons in the alkyl groups, indicating that hydrophobic interaction is a secondary factor for the sequestration of bile acids.
Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.
The acetoacetyl-CoA reductase gene (phbB) of Ralstonia eutropha and the poly[(R)-(-)-3-hydroxyalkanoate] synthase gene (phaCAC) of Aeromonas caviae were introduced into tobacco plant by Agrobacterium mediated transformation method. The resulting transgenic tobacco expressed both introduced genes and the expression of these genes was confirmed by enzymatic analysis and western blotting. GC-MS analysis of the chloroform extract of tobacco leaves indicated that the transgenic plant produced biodegradable polyester, poly-[(R)-(-)-3-hydroxybutyrate]. GPC analysis indicated that the number-average molecular weights (Mn) and polydispersity (Mw/Mn) were 32,000 and 1.90, respectively.
Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of ≥2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.
Twenty-seven Citrus flavonoids were examined for their antiproliferative activities against several tumor and normal human cell lines. As a result, 7 flavonoids were judged to be active against the tumor cell lines, while they had weak antiproliferative activity against the normal human cell lines. The rank order of potency was luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3′,4′,5,6,7,8-heptamethoxyflavone. The structure-activity relationship established from comparison among these flavones and flavanones showed that the ortho-catechol moiety in ring B and a C2-C3 double bond were important for the antiproliferative activity. As to polymethoxylated flavones, C-3 hydroxyl and C-8 methoxyl groups were essential for high activity.
LipL of Pseudomonas sp. strain 109 is a unique lipase capable of catalyzing macrocyclic lactone synthesis using ω-hydroxyfatty acid esters as substrates. Several fatty acid esters were tested as inducers of LipL production. The addition of either soybean oil or a non-ionic detergent (Noigen HC) resulted in a 44 to 45-fold increase in extracellular LipL, and the presence of both resulted in a further 56-fold increase. Among the triglycerides tested, triolein was the most effective, with a 50-fold increase in LipL production. A Northern blot hybridization analysis found that the lipL transcript increased in the presence of soybean oil or Noigen HC, indicating that the production of LipL is regulated at the transcriptional level.
Actin-related proteins (Arps), which are divergent, but apparently homologues to actin, are categorized into 10 classes. While Arps belonging to classes 1-3 were found to be localized in the cytoplasm across eukaryotic phyla, other classes of Arps were found mostly in invertebrates and suggested to contribute to structural modulation of chromatin. Here we report the identification and the characterization of two human isoforms of an Arp not belonging to classes 1-3, which we designated hArpNα and hArpNβ. Both proteins were expressed in HeLa cells and they were found localized within the nucleus. Most interestingly, in different human tissues, hArpNα and β were found to be expressed mutually exclusively, and the expression of hArpNα was absolutely restricted to the brain. These findings suggest that, in vertebrates, members of distantly related Arps might have tissue-specific functions in the nucleus, possibly through structural modulation of chromatin.
Anthocyanin-producing cells in the tepals of flower buds of Hyacinthus orientalis were investigated. In field-grown flowers, anthocyanins were only produced in L2 cells (cells in the sub-epidermis) of tepals. However, when early-stage flower buds were cultured on medium containing GA3, anthocyanins were produced not only in L2 cells but also in L1 cells (cells in the epidermal layer) of tepals. The same anthocyanins were produced in tepals of both field-grown flowers and flower buds cultured in vitro.
The enzymatic synthesis of glucoside compounds using a membrane-associated UDP-glucosyltransferase fraction from Eucalyptus perriniana cultured cells as a water-insoluble catalyst (N. Nakajima, et. al., J. Ferment. Bioeng., 84 (5), pp. 455-460, 1997) has been effectively done by coupling UDPglucose-fermentation by bakers’ yeast. For example, β-thujaplicin (hinokitiol) and p-aminobenzoic acid were converted respectively to their corresponding β-D-monoglucosides with the conversion rate of around 24-26% by the multi-enzymatic system with UDPglucose as a glucose donor, which is produced by yeast cells from glucose and 5′-UMP. Addition of either cellobiose, a substrate of β-glucosidase, or DL-1,2-anhydro-myo-inositol, an inhibitor for the enzyme in the reaction mixture, could increased the yield of these β-D-monoglucosides. This new enzymatic system could also be used for the synthesis of flavonoid glucosides such as isoquercitrin (quercetin 3-O-β-D-glucoside).
It was found that the inhibition of the lysosomal acid lipase activity by rat apolipoprotein A-I (apo A-I) was increased with the degradation of apo A-I by the lysosomal proteases. We demonstrated that apo A-I could effectively inhibit the acid lipase activity even in the presence of the lysosomal proteases using the hepatic lysosomal fraction.
The cDNA encoding a novel three loop protein was cloned from cellular RNA isolated from the venom gland of Bungarus multicinctus multicinctus by RT-PCR. The mature protein has 82 amino acid residues. It shared only 25-38% similarity with some cardiotoxins and did not have sequence similarity with neurotoxins, while its cDNA was about 70% similar to both the cDNAs encoding neurotoxins and the cDNAs encoding cardiotoxins.
For the purification of plant endo-β-N-acetylglucosaminidase, in this report, we introduce a new affinity chromatography using the reduced and carboxymethylated yeast invertase (cm-YI) as a ligand. Two plant endo-β-N-acetylglucosaminidases (endo-LE from tomato fruits (Kimura, Y., et al. Biochim. Biophys. Acta 1381, 27-36 (1998)) and endo-GB from Ginkgo biloba seeds (Kimura, Y., et al. Biosci. Biotechnol. Biochem., 62, 253-261 (1998)) could completely bind to the high-mannose type N-glycans linked to the immobilized yeast invertase and the activities of both enzymes could be recovered by increasing the concentration of NaCl. By using this purification procedure with some other purification procedures, endo-LE could be purified 1,700-fold and endo-GB was purified to apparent homogeneity at 63 kDa as reported previously.
Bovine lactoferrin (LF) and lactoferricin B (LFcin B), an antimicrobial peptide derived from bovine LF, inhibited thiobarbituric acid-reactive substance (TBARS) formation in a iron/ascorbate-induced liposomal phospholipid peroxidation system. The inhibition of TBARS formation occurred with N-acylated 9-mer peptides with a core sequence of LFcin B and, compared to LFcin B, their antioxidant effect was clearly observed at a concentration almost 100 times lower.
The preventive effects of nasunin (delphinidin-3-[4-p-coumaroyl-rhamnosyl(1→6)glucoside]-5-glucoside) on paraquat-induced oxidative stress were determined in rats. Decreased food intake and body weight gain and increased lung weight by feeding the rats a diet containing paraquat were clearly suppressed by supplementing nasunin to the paraquat diet. Paraquat feeding increased the concentration of thiobarbituric acid-reactive substances (TBARS) in liver lipids and the atherogenic index, and decreased the liver triacylglycerol level. These effects were also suppressed by supplementing nasunin to the paraquat diet. In addition, catalase activity in the liver mitochondrial fraction was markedly decreased by feeding the paraquat diet, this decrease being partially suppressed by supplementing the paraquat diet with nasunin. These results suggest that nasunin acted preventively against the oxidative stress in vivo that may have been due to active oxygen species formed through the action of paraquat.
The formation mechanism for the potent antioxidative o-dihydroxyisoflavones, 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), was studied by incubating whole soybeans in a solid culture and a soybean extract in a liquid culture with Aspergillus saitoi. Analyses of changes in the isoflavone analogue content, β-glucosidase activity, and isoflavone hydroxylation ability indicated that 8-OHD and 8-OHG were formed from daidzein and genistein, respectively, by microbial hydroxylation, being respectively liberated from daidzin and genistin by β-glucosidase from A. saitoi during incubation. No selective hydroxylation reaction at the 8-position of daidzein and genistein were apparent during the vegetative stage, but were induced at the stage of sporulation.
Linoleic acid was encapsulated with pullulan, maltodextrin and gum arabic at various weight ratios of the fatty acid to wall material by the hot-air-drying method. The autoxidative process of the encapsulated linoleic acids was observed at 37°C and at a relative humidity of 75%. The weight ratio strongly affected the autoxidative process, autoxidation being more suppressed with smaller ratios. The autoxidation process of encapsulated linoleic acid leveled off at fraction Y∞ of the unoxidized substrate within 15 days. The Y∞ value strongly depended on both the ratio and the wall material, and steeply decreased near the ratio of 0.75 for every wall material. The dependence of the Y∞ value on the weight ratio was analyzed by the two- and three-dimensional models for percolation theory. The two-dimensional model expressed well the experimentally observed dependence.
We studied the effect of dietary vinegar on calcium absorption by using ovariectomized rats fed on a low-calcium diet. The apparent absorption of calcium was higher when the rats were fed on a diet containing 1.6% vinegar for 32 days than when fed on a diet without vinegar (P<0.05). The calcium content in the femur of the rats given diets containing 0.4% and 1.6% vinegar were also higher (P<0.05). The serum parathyroid hormone level was lower and the crypt depth of the duodenum thicker in the rats fed on a diet containing 1.6% vinegar (P<0.05). These results suggest that dietary vinegar enhanced intestinal calcium absorption by improving calcium solubility and by the trophic effect of the acetic acid contained in vinegar, which would reduce the bone turnover caused by ovariectomy and be helpful in preventing osteoporosis.
The antigenic determinants of bovine β-casein (β-CN) were localized by using twenty overlapping peptides encompassing the entire sequence of β-CN and anti-β-CN antisera from outbred mouse, rabbit and goat. The profile of the reactions was characteristic to the species, the dominant antigenic regions being 80-95, 143-158 and 195-209 in mouse, 1-16 in rabbit and 100-115 in goat. Regions 1-16, 100-115, 121-136 and 143-158 were antigenic in all three species. The number of antigenic regions recognized by goat was much fewer than that by mouse and rabbit, possibly because of the homology between bovine and goat β-CN. A mixture of the twenty peptides could absorb about 50-60% of β-CN specific antibodies from each species. Furthermore, the mouse and rabbit anti-β-CN antibodies were also specific to the phosphorylated regions. We therefore conclude that the major antigenic determinants on β-CN would be largely sequential and include the phosphorylated sites.
The acidic fraction of an extract of the culture liquid of Aspergillus repens MA0197 showed strong antioxidative activity when tested by the ferric thiocyanate and TBA methods. Chromatographic purification of this acidic fraction gave an active substance identified as Neoechinulin A. This compound showed higher antioxidative activity than α-tocopherol and could be expected to act as an antioxidant in Katsuobushi.
The effects of medium composition on the production of β-glucosidase (amygdalase and linamarase) by Penicillium aurantiogriseum P35 were studied and the medium optimized as follows (g/l of deionized water): pectin, 10.0; (NH4)2SO4, 8.0; KH2PO4, 8.0; Na2HPO4, 2.8; MgSO4•7H2O, 0.5; yeast extract, 4.0; initial pH 6.0. When grown in a bench fermenter on this medium, the fungus produced 50.5 mU of amygdalase and 9.4 mU of linamarase per ml of culture broth. Two β-glucosidases (PGI and PGII), each having amygdalase and linamarase activities, were recovered from the culture broth and purified; their relative molecular weights, as native enzymes, were estimated to be about 247000 and 147000, respectively. Both enzymes showed the same optimum pH (6.0) but different optimum temperatures (55 and 60°C for PGI and PGII, respectively). Thermostability (10 min at 60°C) and half-life of enzyme activity (7 hours at 60°C) of PGII were higher than those of PGI (10 min at 50°C and 2 hours at 55°C, respectively). A wide range of cyanogenic glycosides (such as tetraphyllin B, epivolkenin, gynocardin, passibiflorin, prunasin, taxiphyllin, amygdalin, lucumin, sambunigrin, dhurrin, linamarin and cardiospermin sulfate) were hydrolyzed by both enzymes.
Intact cells of Thiobacillus ferrooxidans NASF-1 incubated under anaerobic conditions in a reaction mixture containing 0.5% colloidal sulfur produced hydrogen sulfide (H2S) extracellularly. The amount of H2S produced by cells increased corresponding to the cell amounts and colloidal sulfur. Two activity peaks of H2S production were observed at pH 1.5 and 7.5. We tentatively called the enzyme activities pH 1.5- and pH 7.5-sulfur reducing systems, respectively. Seven strains of T. ferrooxidans tested had both the activities of pH 1.5- and pH 7.5-sulfur reducing systems, but at different levels. T. ferrooxidans NASF-1 showed the highest activity of the pH 1.5-sulfur reducing system and strain 13598 from ATCC showed the highest activity of the pH 7.5-sulfur reducing system. Further characteristics of H2S production were studied with intact cells of NASF-1. The optimum temperatures for pH 1.5- and pH 7.5-sulfur reducing systems of NASF-1 were 40°C. Hydrogen sulfide production continued for 8 days and total amounts of H2S produced at pH 7.5 and 1.5 were 832 and 620 nmol/mg protein, respectively. The pH 7.5-sulfur reducing system used only colloidal sulfur as the electron acceptor. However, the pH 1.5-sulfur reducing system used both colloidal sulfur and tetrathionate. Thiosulfate, dithionate, and sulfite could not be used as the electron acceptor for both of the sulfur reducing systems. Potassium cyanide activated by 3- fold the pH 1.5-sulfur reducing system activity at 0.5 mM but did not affect the activity of the pH 7.5-sulfur reducing system. An inhibitor of sulfite reductase, p-chloromercuribenzene sulfonic acid, did not affect either enzyme activity. Sodium molybdate and monoiodoacetic acid strongly inhibited the activity of the pH 1.5-sulfur reducing system at 1.0 mM, but not the activity of pH 7.5-sulfur reducing system.
Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.
The complete sequences of the 18S rRNA gene fragments of the type strains of the cactophilic yeast species, Pichia antillensis, Pichia caribaea, Phaffomyces opuntiae, Phaffomyces thermotolerans, Starmera amethionina var. amethionina, and Starmera amethionina var. pachycereana were determined and compared. The type strain of Phaffomyces opuntiae had two kinds of the 18S rRNA gene sequences of which base differences were counted to be 15 and of which the percent similarity was calculated to be 99.1. The type strains of P. antillensis, P. caribaea, and Starmera amethionina var. pachycereana had the Q-7 system. The phylogenetic analyses showed that the genera Phaffomyces and Starmera were monophyletic and distant from each other and from the other species examined of the ascogenous teleomorphic genera, and that P. antillensis and P. caribaea were included within the clusters of the genera Phaffomyces and Starmera, respectively. The two Pichia species were transferred to the genera Phaffomyces and Starmera as the new combinations, Phaffomyces antillensis and Starmera caribaea. The new family Phaffomycetaceae was proposed as the type genus Phaffomyces.
An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast. To obtain a stable reversionless host strain, we constructed an S. exiguus strain carrying leu2::ScURA3 by disrupting the S. exiguus LEU2 gene with the S. cerevisiae URA3 gene. A vector plasmid unique to S. exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S. exiguus into an Escherichia coli phagemid, pUC119. The vector constructed, pTH119 was able to transform the S. exiguus leu2::ScURA3 strain to Leu+ efficiently. The stability of the vector in the S. exiguus host cells resembled that of a YRp-type vector in S. cerevisiae.
An energy-dependent K+/H+ antiport system is found in Enterococcus hirae ATCC 9790 cultured in a standard complex medium (Y. Kakinuma, and K. Igarashi, J. Biol. Chem. 263:14166-14170, 1988). We have now found that the activity of this antiport system was totally missing in cells cultured in a defined medium. In this defined medium, E. hirae did not grow well at pH near 9, but grew normally at pH below 7.5. This antiport system is important at high pH but dispensable at lower pH for ion homeostasis of this bacterium.
Appressorium formation in germinating conidia of Magnaporthe grisea was inhibited on inductive and on noninductive surfaces by monounsaturated fatty acids with chain lengths of 16, 18, or 20 carbon atoms. On a noninductive surface, the inhibition was only observed upon stimulation with 1,16-hexadecanediol or oleyl alcohol, but not upon stimulation with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. In the C18-series, fatty acids with a double bond in position 9 were the most active ones. At 1 μg/ml of oleic or elaidic acid, less than 30% of the germinated conidia formed appressoria. The mode of inhibition was competitive to the inducing agent. On an inductive surface, compared to a noninductive surface the concentrations of oleic and elaidic acid needed for inhibition of appressorium formation were one order of magnitude higher. Methyl esters of inhibitory fatty acids and acids with two double bonds were not active. Like oleyl alcohol, elaidyl alcohol and petroselinyl alcohol stimulated infection structure formation on the noninductive surface.
Alkaliphilic Bacillus sp. C-125 was taxonomically characterized by physiological and biochemical characteristics, 16S rDNA sequence similarity, and DNA-DNA hybridization analyses. Phylogenetic analysis of strain C-125 based on comparison of 16S rDNA sequences showed that this strain is closely related to Bacillus halodurans. DNA-DNA hybridization analysis was done comparing C-125 and related Bacillus reference strains. The highest level of DNA-DNA relatedness (86%) was found between strain C-125 and B. halodurans. Our findings demonstrate that strain C-125 is a member of the species B. halodurans.
The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant. With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.
p. 592, right column, line 13, anti-asthmatic
p. 592, right column, line 10 from the bottom, thiosulfinates
p. 593, right column, line 21 and 23, and in Table 1, μg/ml