ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO·) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO· at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.
The glucosidic precursor of marmelo lactones was synthesized by employing a common intermediate which had been used for the synthesis of the glucosidic precursor of marmelo oxides. The synthesis was performed by modifying the former procedure. Monochloroacetyl was adopted to protect both the glucose and aglycon hydroxyl groups for selective transesterification in the presence of the glycosyl ester. Glycosylation of the aglycon carboxyl group with 1-α-bromopermonochloroacetylglucose and final selective alcoholysis yielded the target glucoside.
Cucumber (Cucumis sativus L.) roots were analyzed by HPLC and TLC for their levels of secondary metabolites upon inoculation with the arbuscular mycorrhizal fungus, Glomus caledonium. Three compounds in EtOAc extracts from the mycorrhizal roots showed significant increases six weeks after inoculation. These compounds were isolated by column chromatography and determined to be two novel triterpenes, 2β-hydroxybryonolic acid (2β,3β-dihydroxy-D:C-friedoolean-8-en-29-oic acid) and 3β-bryoferulic acid [3β-O-trans-ferulyl-D:C-friedooleana-7,9(11)-diene-29-oic acid], and the known triterpene, bryonolic acid, by spectroscopic methods. Time-course experiments showed that the levels of the three terpenoids in cucumber roots were significantly increased by the application of a 53-μm sieving from a soil inoculum of the arbuscular mycorrhizal fungus containing soil microbes but no mycorrhizal fungi, and that mycorrhizal colonization further promoted the terpenoid accumulation. Inoculation with Glomus mosseae also enhanced the accumulation of the triterpenes, whereas no accumulation was observed by inoculating with the fungal pathogen, Fusarium oxysporum f. sp. cucumerinum. 2β-Hydroxybryonolic acid was also isolated from the roots of melon and watermelon.
Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced α-amylase induction in embryoless barley seeds at 120 μM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 μM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.
A new sesquiterpene lactone (1) was isolated with known dihydrochrysanolide derivatives (2 and 3) from the flowers of Chrysanthemum coronarium L., and their structures were identified by spectroscopic data. The stereochemistry of the epimers (1 and 2) was determined from NOESY data and an X-ray crystallographic analysis. The isolated compounds (1–3) were examined for their cytotoxic activity against such human cell lines as A549, PC-3 and HCT-15.
(1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), a potent agonist of metabotropic glutamate receptors, was synthesized from L-serine. The chiral quaternary center was constructed by C–H insertion of the alkylidenecarbene, this being generated by the reaction between lithiotrimethylsilyldiazomethane and the corresponding ketone.
The effect of a revised Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.). The effects of a high iron concentration and low concentration of zinc on betacyanin production were not cumulative. The composition of the new revised medium for high betacyanin production was established by reducing the concentration of inorganic nitrogen (30 mM), modifying the ratio of ammonium to nitrate (1:14), reducing the concentration of zinc (0.0003 mM), and removing copper and cobalt. The revised LS medium enabled the maximum betacyanin yield of 550 mg/l to be obtained from a 14-day culture. This medium promoted the betacyanin production in three types of cell line differing in the betacyanin productivity. The betacyanin productivity (40 mg/l•day) was higher than that quoted in any other previous reports.
Two polyhydroxysteroids and three steroidal saponins (pectiniosides A, B and C) were isolated as bioactive substances from Asterina pectinifera. These compounds inhibited the release of guinea-pig skin stratum corneum cells by 48–67% (control, 0%) at 2 mg/ml. It is suggested that the bioactivity of these compounds is connected with the remedial and preventive effects of an aqueous extract of A. pectinifera on rough human skin.
A perilla seed (Perilla frutescens Britton var. japonica Hara) extract was examined for its antimicrobial activity against oral cariogenic streptococci and periodontopathic Porphyromonas gingivalis. Luteolin, one of the components of perilla seed, showed the strongest antimicrobial effect among the phenolic compounds. According to our results, perilla seed may be the source of an antimicrobial agent that could prevent dental caries and periodontal diseases.
NADH oxidation by manganese peroxidase (MnP) was done in a reaction mixture including either α-hydroxy acid or acetate. The oxidation in the former reaction mixture was inhibited by a catalase and was accelerated by exogenous H2O2, while the oxidation in the latter reaction mixture was inhibited by a superoxide dismutase and was not accelerated by the exogenous H2O2. These results indicated that there are significant differences between the two reaction systems, particularly, in the active oxygen species involved in the reactions. Additionally, the experiment of MnP reduction with Mn(II) suggests that MnP has a separate catalytic activity other than an oxidation of Mn(II) to Mn(III) in the reaction mixture including acetate.
Secretory phospholipases A2 (PLA2) from Naja naja naja (cobra snake) venom, from Bothrops neuwiedii (crotalid snake) venom (two isoforms) and from bee venom were modified with tresylated monomethoxy poly(ethylene glycol) (TMPEG). The kinetic and inflammatory properties of the adducts (PEG-PLA2) were measured. As found by gel permeation chromatography, 95-100% of P-1 PLA2 from B. neuwiedii and PLA2 from N. naja naja venom change their chromatographic mobility after TMPEG treatment. By contrast, only 50-60% of both P-3-PLA2 from B. neuwiedii and PLA2 from bee venom modify their elution profile from Superdex 75.
All the modified proteins preserved the enzymatic activity toward phospholipid monolayers, but with a reduced specific activity and greater lag times than the unmodified controls. These results suggest that the PEG-PLA2 complexes would have an altered interaction with lipid membranes. The PEG-linked proteins preserve their edema-inducing activity evaluated by the rat hind-paw edema test except for N. naja naja PEG-PLA2 in which inflammatory activity was significatively decreased. Altogether, the results show a partial dissociation of catalytic and inflammatory activities of Group II and III secretory PLA2s after their modification with PEG.
A psychrophilic alkaline phosphatase (EC 220.127.116.11) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246–2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single α-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.
A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N′-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.
The effects of Pi deficiency on gene expression in Pholiota nameko were examined. A cDNA library was constructed from poly(A)+ RNA isolated from mycelia cultured in Pi-depleted (P−) media, and 150 clones corresponding to Pideficiency-inducible (pdi) genes were selected by differential hybridization with probes prepared from poly(A)+ RNAs from the mycelia cultured in the Pi-supplied (P+) and P− media. These clones were considered to derive from 31 genes by cross-hybridization. Northern blot analysis showed that these pdi genes were expressed in various patterns during Pi deficiency. Among the clones, the DNA sequences of pdi85 and pdi343 were analyzed. The deduced amino acid sequences indicated that they have structural similarities to Pi and metabolite transporters.
A novel β-glycosidase-producing microorganism was isolated from soil and identified as Aspergillus fumigatus AP-20 based on its taxonomical characteristics. The enzyme was found to be an extracellular protein in the culture of the isolated fungus and was purified 88-fold by fractionation with ammonium sulfate followed by successive column chromatographies on phenyl-Sepharose HP and Mono P HR. The molecular mass was estimated to be 47 kDa by SDS-PAGE and the isoelectric point to be pH 6.0 by isoelectric focusing. The purified enzyme was highly specific for a substrate, p-nitrophenyl β-primeveroside (6-O-β-D-xylopyranosyl-β-D-glucopyranoside), which was cleaved in an endo-manner into primeverose and p-nitrophenol.
A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the β1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having α1-3 fucose and β1-2 xylose residues (GlcNAc2~0Man3Xyl1Fuc1~0GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man9~5GlcNAc2).
We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 μg/ml), nearly 95% of the cells had apoptotic nuclear morphological changes, while 53%, 30%, and 10% of the cells showed apoptotic changes after exposure to ricin (0.1 μg/ml), diphtheria toxin (10 μg/ml), and cycloheximide (10 μg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis.
The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50–90°C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4–150 μmol•mg−1•mM−1) along with the increase of temperature from 50 to 90°C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.
We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.
Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75°C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3°C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.
We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues. 5-Bromo-2′-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells.
We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively. In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity. RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I). Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU. Furthermore, kinetic parameters for RNase NW (Km, 0.778 mM and kcat, 1938 min−1) and RNase NGR3 (Km, 0.548 mM and kcat, 408 min−1) were calculated using GpU as a substrate.
Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe α-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an α-glucosidic linkage from β-glucosyl fluoride and 4-nitrophenyl (PNP) α-glucoside to produce two kinds of PNP α-diglucosides, α-isomaltoside and α-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP α-isomaltoside and α-maltoside, respectively. PNP monoglycosides, such as α-xyloside, α-mannoside, or β-glucoside, acted as the substrate, but PNP α-galactoside and maltose could not. No detectable product was observed in the combination of α-glucosyl fluoride and PNP α-glucoside. This study is the first report on an “α-glycosynthase”-type reaction to form an α-glycosidic linkage.
Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5′-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.
We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of α-amino-β-carboxymuconate-η-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan- niacin metabolism.
The effects of dietary powdered green tea (PGT) and theanine on in vivo hepatoma growth and cancerous hyperlipidemia were investigated in rats that had been implanted with a rat ascites hepatoma cell line of AH109A cells. The hepatoma-bearing rats were fed with a 20% casein diet (20C), 20C containing 2% PGT, or 20C containing 0.1% theanine for 14 days. Dietary PGT significantly and time-dependently reduced the solid tumor volume and weight as did dietary theanine. The hepatoma-induced endogenous hyperlipidemia, which was characterized by rises in the serum cholesterol (hypercholesterolemia) and triglyceride (hypertriglyceridemia) levels, was significantly suppressed by PGT and theanine supplementation. Bile acid excretion into the feces was significantly higher in the PGT- and theanine-fed rats than in the control rats. This inhibition of hypercholesterolemia may have resulted from tumor growth suppression as well as increased excretion of steroids from the body. These results suggest that PGT had both anti-proliferative activity toward hepatoma cells and hypolipidemic activity in the hepatoma bearing rats. They also suggest that theanine was, at least in part, responsible for the PGT actions.
The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 μM of LPC, the taurine uptake was rapidly decreased by approximately 60%. This decrease was accompanied by an increase in the Km value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.
The application of ω-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry.
In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20%(w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70°C. A plot of the natural logarithm of the frequency factor, ln ka0, vs. the activation energy, Ea, demonstrated that ln ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.
The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (−)-epigallocatechin gallate (EGCg) were kinetically analyzed. The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission. Coordinated EGCg accelerated these reactions. Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl− ions and EGCg added. Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one. This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.
Rats were fed on a diet containing 0.5% cholesterol oxidation products (oxysterols) or 0.5% cholesterol for 30 min, and their lymph was collected for 7 h. The amount of each of the individual oxysterols absorbed in the lymph depended on the ingested amounts, but the recovery was the highest for 5α,6α-epoxycholesterol (10.5%), this being followed by 7-ketocholesterol (5.8%), cholestanetriol (5.2%), 7β-hydroxycholesterol (4.8%), 7α-hydroxycholesterol (3.4%), 5β,6β-epoxycholesterol (2.2%), and 25-hydroxycholesterol (1.8%). A diet enriched with oxysterol, but not cholesterol, resulted in increased transport of triacylglycerols in the lymph. These results suggest that the absorption rate of oxysterols depends on the type, and indicate that the effect of dietary oxysterols on the lymphatic transport of triacylglycerols differs from that of dietary cholesterol. It therefore remains to be determined which oxysterol was responsible for the triacyglycerol transport.
Mice were fed with swine gastric mucin in a basal diet for 5 weeks. In 5 week-old mice, a 2% mucin diet significantly decreased nitric oxide levels of serum and liver. Reduction of serum total cholesterol and triglyceride and increase of HDL-cholesterol level were also significant with the mucin diet. In 14 month-old mice, the mucin diet was less effective.
The inhibitory effect of an onion extract on browning of potato was investigated. The addition of the heated onion extract to potato exhibited a marked inhibitory effect on potato polyphenol oxidase and the formation of a brown color. The inhibitory effect of the onion extract was dependent upon its heating temperature. The addition of both glycine and glucose increased the inhibitory effect of the onion extract toward potato polyphenol oxidase.
An onion extract possessed anticoagulative activity apparently by inhibiting thrombin, and it was dependent upon the concentration of onion extract added. Boiling the onion extract at 100°C for 30 min made no difference to its anticoagulative activity compared to that of fresh onion. The anticoagulative activity of the onion extract was also retained after an acid treatment by incubating at pH 2.0 for 4 hrs. However, dialyis of the extract substantially eliminated the anticoagulative activity. Therefore, the anticoagulant in the onion extract seems to have been a heat- and acid-stable substance of low molecular weight.
New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of Nε-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys.
In order to discover the effect of CLA on the body fat size and serum cytokine levels, four groups of male mice were fed diets containing either 1% linoleic acid (LA) or conjugated linoleic acid (CLA) with or without 0.2% sesamin for 8 weeks. The weight gain and feed efficiency were significantly lower in the CLA groups. CLA significantly reduced relative weights (g/100 g body weight) of epididymal and perirenal adipose tissues, in particular the former. Concentrations of serum TNF-α and leptin were significantly reduced by dietary CLA. Sesamin did not show additional effects in all of these parameters. There was a positive correlation between cytokine production and body-fat reducing potential of CLA. These results indicated that mice appeared to be a hyperresponder to dietary CLA insofar as the reduction of body fat size is concerned.
Distinct enzyme activities were found in extracts from Stenotrophomonas maltophilia showing dye-linked oxidation of polypropylene glycols. The activities were induced when polypropylene glycols served as sole carbon and energy sources for the bacterium. In the logarithmic phase, most of the enzyme activities (88%) were found in the cytoplasm. In the stationary phase, more than half of the activities (54%) were found on the membrane, but significant activities were also distributed in the periplasm (34%) and the cytoplasm (12%). The enzyme activities differed from each other in their localization, time of induction in the growth cycle, specificity toward electron acceptor, and electrophoresis mobility.
Acetobacter strains able to produce a thick pellicle at 37°C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37°C or 40°C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30°C than at 37°C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.
Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the nonmevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs 1 and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.
The ice-nucleating bacterium Pantoea ananas KUIN-3 accumulated glucose in cells following a shift in temperature (10°C) from the optimum growth temperature (30°C). This accumulation might be caused by the activation of glucose-6-phosphatase. Although this strain after culturing at 30°C was harmed by freezing, the cryotolerance of this strain was reached about 80% after cold acclimation at 10°C.
Formation of new lipoaminopeptides, acremostatins A, B, and C, was observed during co-cultivation of Acremonium sp. Tbp-5 and Mycogone rosea DSM 12973. Thus, co-cultivation of microorganisms producing related products could be suggested as a suitable way towards diversification of microbial structures.
Med was found as a positive regulator for comK, a master regulator for late competence genes. It was found by Western analysis that the ComK level was decreased in a med mutant. Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med. The results obtained are consistent with the localization of Med at the cell surface. An implication of the cell-surface localization of Med is discussed in terms of comK regulation.
Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon. Cat proteins of strain CA10 were very similar to those of P. putida, although the relatedness in non-coding regions was not high. It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.
The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.