The crystal structures of chitosan-L- and D-lactate salts were studied by X-ray diffraction measurements on fiber diagrams. In each lactate, chitosan took on a different crystalline polymorph depending on the preparation temperature. At low temperature, they gave a similar fiber pattern to that of the type II salt which has been found to be one of the two forms of chitosan acid salts in which the backbone chitosan molecules take on an eight-fold helix. At high temperature, however, the fiber pattern was that of the type I salt, another form of chitosan salt in which the backbone chains apparently retain the 21 symmetry of chitosan itself. The high-temperature polymorph of the L-lactate was a monoclinic (pseudoorthorhombic) unit cell whose lattice parameters were a=10.51, b=10.85, c(fiber axis)=10.34 Å and γ=90°. That of the D-lactate was also a monoclinic cell having parameters a=11.20, b=11.60, c(fiber axis)=10.38 Å and γ=93.0°. Their unit cell volumes coupled with their observed density values indicate that two chains of chitosan lactate were accommodated in each unit cell, that the L-lactate was an anhydrous crystal, but that the D-lactate was hydrated. The preparation temperature at which the salt changed from type II to type I was different between the D- and L-lactate, suggesting that these acids had different affinity to the chitosan molecule. When chitosan powder was suspended in a racemic lactic acid solution, the resultant solution always showed a minus sign for the rotation angle, indicating that D-lactic acid had higher affinity to chitosan than the L-isomer.
This study for the first time achieved an emission spectrometric analysis of the chemiluminescence of flavonoids in the presence of hydrogen peroxide, acetaldehyde and horseradish peroxidase, and revealed that the maximum emission wavelengths (Emax) strictly differ among catechins (Emax 630 nm), theaflavins (Emax 690 nm) and anthocyanins (Emax 675 nm) according to their chemical structures. This technique enabled the direct incorporation of dietary tea catechin into rat intestinal mucosal cells to be spectrometrically confirmed.
The fungal pathogen Septoria spp., that had been isolated from the infected leaves of leafy spurge, produced two phytotoxic compounds that were identified by X-ray diffraction and spectroscopic methods as 1 and 2.
Methionine was subjected to a flame-induced reaction in water or in an aqueous formic acid solution by using a hydrogen (50%)-oxygen (50%), hydrogen (87%)-oxygen (13%) and hydrogen diffusion flame. Besides the already-known stepwise oxidation by a hydroxyl radical, the contribution of a hydrogen atom from the flame to the reaction was recognized when the hydrogen-rich mixtures were employed. Homoserine was obtained under all the reaction conditions employed here, and glutamic acid when employing aqueous formic acid as a solvent. A common intermediate, the 3-carboxy-3-aminopropyl radical, appeared to exist in the reaction pathway. A coupling reaction of this radical with a hydrogen atom, hydroxyl radical and hydroxycarbonyl radical afforded 2-aminobutyric acid, homoserine and glutamic acid, respectively. Lanthionine and S-methylcysteine underwent the same reactions. Increasing the hydrogen content of the fuel and adding formic acid to the solvent resulted in retarding the reaction rate. The latter modification of the reaction system also brought about greater stability of the reaction products.
In our screening program for antioxidants with DPPH radical scavenging activity, we isolated four yellowish compounds from the fermentation broth of Trichoderma sp. USF-2690 strain isolated from a soil sample: two were novel compounds designated bisorbicillinol (1) and demethyltrichodimerol (2), and two were known compounds bisvertinolone (3) and trichodimerol (4). The structures of 1 and 2 were elucidated by spectroscopic evidence and chemical modification. Two compounds seemed to be the oxidized dimers of sorbicillin. In the evaluation of DPPH radical scavenging activity, bisorbicillinol gave the lowest ED50 value (31.4 μM) among the four compounds, equal to that of BHT (27.0 μM).
Coronafacic acid (1) is an acid component of coronatine, and has been isolated from several pathovars of Pseudomonas syringae. Syntheses of C6-non- and C6-alkyl-substituted analogues of 1 were accomplished via intramolecular 1,6-conjugate addition as the key step. Among them, 1 and four C6-alkyl-substituted analogues exhibited potato tuber-inducing activity, but the C6-non-substituted analogue did not. It was revealed that a certain length of the C6-alkyl group was necessary to exhibit activity.
Synopsis: 3-Oxa-OPC and 2-fluoro-OPC homologues which do not undergo β-oxidation were synthesized from esters of odd-numbered OPC homologues by a short-step procedure. The 3-oxa-OPC homologues were synthesized via etherification of an alcohol with tert-butyl bromoacetate under phase-transfer conditions. The 2-fluoro-OPC homologues were synthesized via the addition of the trichloromethyl anion to an aldehyde and subsequent fluorination.
Stereoselectivity in the Michael addition of (Me2C=CH)2CuMgBr and (Me2C=CH)2CuLi to 3-alkyl/H-4-[(tert-butoxycarbonyl)alkyl]-2-cyclohexenone was studied. (Me2C=CH)2CuMgBr showed stereoselectivity in all cases (3-H, Me: anti; 3-n-Bu: syn). This stereoselectivity disappeared in the reaction of (Me2C=CH)2CuLi with 4-[(tert-butoxycarbonyl)methyl]-3-butyl-2-cyclohexenone. However, the stereoselectivity was recovered by elongating the 4-alkyl chain of 2-cyclohexenone to show the same selectivity as that of (Me2C=CH)2CuMgBr.
Okaramine G (1), a new okaramine congener, was isolated from Penicillium simplicissimum ATCC 90288. The structure of 1 was determined by a spectroscopic investigation. Okaramine G exhibited insecticidal activity against silkworms.
A wild type of the Gram-positive bacterium, Bacillus brevis, reduced polycyclic aromatic compounds such as 9-fluorenone to the corresponding alcohol, 9-hydroxyfluorene, at 30°C in an anaerobic atmosphere in a 97% yield by extraction with an organic solvent. The products could be also continuously isolated by dialysis from a flowing reaction solution.
Electrophilic additions of DL- and L-Cys to propenoic acid afforded (RS)- and (R)-2-amino-3-(2-carboxyethylthio)propanoic acids [(RS)- and (R)-ACE], respectively. (RS)-ACE was found to exist as a conglomerate based on its melting point, solubility, and infrared spectrum. (RS)-ACE was optically resolved by preferential crystallization to yield (R)- and (S)-ACE. The obtained (R)- and (S)-ACE were efficiently recrystallized from water, taking account of the solubility of (RS)-ACE, to give them in optically pure form.
Information about the specificity of glycosidase enzymes is important since it affects their use for characterization and synthesis of oligosaccharides. Two α-mannosidases (EC 126.96.36.199), I and II, were isolated from rice beans (Vigna umbellata). The native molecular weight of both isozymes was estimated to be 329,000, but pIs of form I were 5.03-5.34 and pIs of form II were 5.46-6.20. The two isozymes were characterized in terms of optimal pH and temperature, effects of metal ions, inhibition by swainsonine and 1-deoxymannojirimycin, and kinetic parameters for p-nitrophenyl-α-D-mannopyranoside and Manα(1-2)Man. Both enzymes were more specific towards Manα(1-2)Man in both hydrolysis and synthesis, but their hydrolytic specificities towards Manα(1-3)[Manα(1-6)]Man were different.
Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO2 production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system. The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.
The effects of O-glycosylation inhibitors on the growth and differentiation of the human acute promyeloblastic leukemia cell line HL-60 were studied to examine whether the O-glycosylation is needed for HL-60 cells to differentiate into granulocyte-like cells or monocyte-macrophage-like cells. N-Acetyl-α-D-galactosaminides, inhibitors of mucin-type oligosaccharide synthesis, and N-acetyl-β-D-galactosaminides did not affect either growth or differentiation. β-D-Xylosides, the artificial initiators of glycosaminoglycan synthesis, also were tested. Only 4-methylumbelliferyl-β-D-xyloside induced HL-60 cells, to differentiate, and they differentiated into granulocyte-like cells, assessed by reduction of nitroblue tetrazolium, Giemsa staining, and esterase double-staining. The aglycon portion of 4-methylumbelliferyl-β-D-xyloside, 4-methylumbelliferone, caused the differentiation. Thus we could find a new drug that induces the differentiation of HL-60 cells.
A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant foodstuffs.
Purine nucleoside phosphorylase (PNP) was purified to homogeneity. The molecular weight of the enzyme was 170,000. The enzyme consisted of six subunits, each with a molecular weight of 27,000. Serratia PNP had ten times the affinity for adenosine and deoxyadenosine than for inosine and deoxyinosine in a pattern characteristic of bacterial PNP. 1-Methylinosine and 1-methylguanosine, which have no affinity for mammalian PNP, bound Serratia PNP. In terms of kcat/Km, the substrate specificities were in the descending order of guanosine, inosine, and adenosine. When inosine or deoxyinosine was used as a variable substrate, a biphasic reciprocal plot with upward curvature was observed. The values of the Hill coefficient were 1.2 and 1.1 for inosine and deoxyinosine, respectively. Positive cooperativity seemed to be involved in the binding of inosine and deoxyinosine to the enzyme.
Precise substrate specificities of α-L-arabinofuranosidases from Aspergillus niger 5-16 and Aspergillus niger (Megazyme) were investigated. Both enzymes hydrolyzed arabinan and debranched-arabinan at almost the same rate. The α-L-Arabinofuranosidase from A. niger (Megazyme) preferentially released arabinosyl side-chains of arabinan. The enzyme tore off both arabinoses attached to O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose and O-β-D-xylopyranosyl-(1→4)-[O-α-L-arabinofuranosyl-(1→3)]-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose, but did not tear off xylosyl-arabinose from O-β-D-xylopyranosyl-(1→2)-O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose. The enzyme from A. niger (Megazyme) hydrolyzed methyl 2-O-, methyl 3-O- and methyl 5-O-α-L-arabinofuranosyl-α-L-arabinofuranosides to arabinose and methyl α-L-arabinofuranoside in the order of (1→5)->(1→2)->(1→3)-linkages. On the other hand, α-L-arabinofuranosidase from A. niger 5-16 successively liberated the arabinose of arabinan from non-reducing terminals. The enzyme hydrolyzed in the order of (1→2)->(1→3)->(1→5)-linkages. Both of the enzymes hydrolyzed the (1→3)-linkage more than the (1→5)-linkage of methyl 3,5-di-O-α-L-arabinofuranosyl-α-L-arabinofuranoside.
As part of a study of the mechanism by which Bacillus thuringiensis insecticidal crystal protein acts, a Bombyx mori receptor to the CryIA(a) toxin specific for lepidopterans was examined. Histological examination showed that the toxin acted on the brush-border membrane of the midgut columnar cells and broke its infolding structure, causing cell lysis. The membrane vesicles were purified, and a 175-kDa protein binding the toxin was found that accounted for some 0.015% of membrane proteins. The protein, designated BtR175, was a glycoprotein that reacted with concanavalin A. Anti-BtR antibodies inhibited the binding of toxin to membrane vesicles in vitro and decreased the effect of the toxin to silkworms in vivo. BtR175, although found in the gut, was not found in fat bodies, integument, or silk glands. These results indicated that BtR175 was the receptor protein for the insecticidal toxin. Proteins (137 and 107 kDa) binding the CryIA(a) toxin also were found in the gut membranes of Tenebrio moritor larvae, a coleopteran not sensitive to the toxin. The specificity of the toxin could not be explained only in term of the existence of its binding protein.
Bacillus thuringiensis strains produce insect-specific Bt toxins. Bt CryIA(a) toxin binds to a 175-kDa glycoprotein (BtR175) on the microvillus membranes of columnar cells in the Bombyx mori midgut and causes lysis of the cells. BtR175 was purified, and its cDNA was cloned. The cDNA encodes a newly identified 193.3-kDa preproprotein form of BtR175 that includes nine extracellular cadherin repeats, a 23.5-kDa membrane-proximal domain, a membrane-spanning region, and a 13.6-kDa cytoplasmic domain. Spodoptera frugiperda cells transfected with a recombinant baculovirus DNA carrying the cDNA produced a 175-kDa protein that reacted with anti-BtR antibodies and the Bt CryIA(a) toxin.
Catechol 2,3-dioxygenase (C23D; EC 188.8.131.52) was purified to homogeneity from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and the purified C23D was characterized. The molecular mass estimated by gel filtration was 110 kDa. The enzyme dissociated into four identical subunits each with the molecular mass of 33 kDa. The enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, in optimal pH for catechol, extradiol cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical properties. The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp. AW-2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase from toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1% identical to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity for 3-methylcatechol, these results suggest that the differences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity.
We isolated a cDNA encoding catalase from green pepper seedlings elicited with arachidonic acid, based on the amino acid sequences of the purified protein. The nucleotide sequence of the isolated cDNA contained a single open reading frame predicted to encode 492 amino acid residues with a calculated molecular mass of 56439.0 daltons. The deduced amino acid sequence contained the amino acid sequences found by sequencing of the peptides. The total deduced amino acid sequence showed high similarity with those of the other plant catalases reported so far and was found to possess the peroxisomal targeting sequence conserved among plant catalases. Transcription of the catalase gene in green pepper seedlings was found to be induced by treatment with arachidonic acid.
Triacylglycerol lipase (L3) was purified from Aspergillus oryzae RIB128 by ammonium sulfate fractionation, acetone precipitation, anion-exchange chromatography, and gel filtration. The purified enzyme was formed from a glycoprotein and a monomeric protein with molecular masses of 25 and 29 kDa, by SDS-PAGE and gel filtration, respectively. The optimum pH at 40°C was 5.5 and the optimum temperature at pH 5.5 was 40°C. The enzyme was stable between a pH range of 4.0-7.5 at 30°C for 24 h, and at up to 30°C at pH 5.5 for 1 h. Heavy metal ions, detergents, DFP, and DEP strongly inhibited the enzyme activity. The lipase hydrolyzed not only triacylglycerols but also monoacylglycerols and diacylglycerols. The enzyme had higher specificity toward triacylglycerols of middle-chain saturated fatty acids than short-chain or long-chain fatty acids. The enzyme had 1,3-positional specificity. The N-terminal amino acid sequence of the enzyme was not significantly similar to that of other lipases with published sequences.
We have reported a simple and rapid method to discriminate species in the genus Pleurotus by analysis of restriction-fragment-length polymorphism of whole-cell DNA, and found that several restriction enzymes gave DNA bands useful in such discrimination, but other enzymes tested did not. In the present study, we report the reason why there were useful and useless enzymes; the effective enzymes digested rDNA into small fragments that did not interfere with the detection of DNA bands useful for discrimination. The origin of these discriminative DNA bands was found to be mitochondrial DNA when the banding profiles of whole-cell DNA, mitochondrial DNA, and nuclear DNA were compared. Consequently, our method could be used for rapid and simple identification of mitochondrial DNA type in the genus Pleurotus. The results were used to study mitochondrial inheritance, and we found that only the nucleus but not the mitochondria migrated during the mating of Pleurotus cornucopiae with P. citrinopileatus.
A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate reductase (DHFR) was used to search for a proteolytic site that gave new N- and C-termini on circularized DHFR with enzyme activity. Of the six site-specific proteolytic enzymes tested, three proteases, Achromobacter protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and Staphylococcus aureus V8 protease, cleaved a single site of the circularized DHFR to form circular permuted variants. Twenty-four possible sites for cleavage were found formation of eight circular permuted variants was suggested by results of N-terminal sequence analysis of the linearized proteins isolated by gel filtration in the presence of 5 M guanidine hydrochloride. Mapping of the predicted cleavage sites on the DHFR molecule suggested that they were not all at a specific loop and, therefore, there are many possible circular permuted variants.
Trehalose phosphorylase was purified from the cell extracts of Catellatospora ferruginea. The enzyme had an apparent molecular weight of 400,000 by gel filtration and 98,000 by SDS-PAGE, suggesting that the enzyme was a tetramer. The enzyme was specific for trehalose in phosphorolysis and specific for β-D-glucose 1-phosphate in synthesis. In addition to D-glucose, D-xylose and D-fucose were also possible sugar acceptors during synthesis. Phosphate ions were a key to the activity and stability of the enzyme, controlling the equilibrium of the reversible reaction and the heat stability of the enzyme. The enzyme was strongly inhibited by p-chloromercuribenzoate and pyridoxal phosphate. The enzyme was inactivated by heat or by storage frozen with ammonium chloride and lithium chloride.
To characterize the pharmacological effects of N-tert-butyl-α-phenylnirone (PBN) on life span, we administered PBN in drinking water to 24.5-month-old mice, and the survivors were counted. Their water consumption and body weights were measured as biological markers. PBN-treated animals as compared with control animals had prolonged mean and maximum life spans. Their water consumption decreased but no significant change was found in their body weights, indicating that the metabolism was improved. Results showed that PBN indeed affects physiological functions and extends life span. We propose that nitric oxide release from PBN may be involved in altering the aging process.
In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity. We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.
Two isoforms, F1 (pI 4.7) and F2 (pI 4.9), of α-fucosidase were purified from the viscera of Pomacea canaliculata. The thermostability of F1 was higher than that of F2. Both were 260-kDa proteins containing polypeptides (55 and 52 kDa) and sugar. There were small differences between F1 and F2 in the optimum conditions for the enzyme reaction and pH stability.
We found that paratropomyosin bound to β-connectin, in examining binding of paratropomyosin at the junction of A- and I-bands of sarcomeres. The turbidity of a mixture of β-connectin and paratropomyosin was greater with more paratropomyosin added, but high concentrations of Ca2+ suppress this increase. These results suggest that paratropomyosin is released from connectin filaments at the A-I junction region by increased concentrations of calcium ions in postmortem skeletal muscles.
The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.
The effects of dietary α-linolenic, eicosapentaenoic and docosahexaenoic acids on the enzyme activities related to hepatic lipogenesis and β-oxidation were compared under constant polyunsaturated/monounsaturated/saturated fatty acids and n-6/n-3 ratios of dietary fats in rats. Dietary fat containing linoleic acid as the sole polyunsaturated fatty acid (PUFA) was also given as a control. The concentration of serum triglyceride and phospholipid in the three n-3 PUFA groups was lower than in the linoleic acid group. The hepatic triglyceride concentration was lower and the phospholipid concentration was higher in the three n-3 PUFA groups than in the linoleic acid group. Cytosolic fatty acid synthase (FAS) activity was lower in the n-3 PUFA groups than in the linoleic acid group, the reduction being more predominant in the eicosapentaenoic acid and docosahexaenoic acid groups than in the α-linolenic acid group. The cytosolic activities of the NADPH-generating enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and the malic enzyme, were lower in the three n-3 PUFA groups. The activity of carnitine palmitoyltransferase (CPT) in mitochondria was higher only in the eicosapentaenoic acid group than in the other groups. The activity of Mg2+-dependent phosphatidate phosphohydrolase (PAP) in microsomes and cytosol was lower in the eicosapentaenoic and docosahexaenoic acid groups than in the linoleic acid group, while there was no effect of dietary fats on the activities of diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (G3PAT) in microsomes. The CTP: phosphocholine cytidylyltransferase (CT) activity in the homogenate was lower in the n-3 PUFA groups, the reduction being more prominent in the eicosapentaenoic and docosahexaenoic acid groups than in the α-linolenic acid group. The choline kinase (CK) activity in cytosol was lower in the eicosapentaenoic acid group than in the linoleic acid group. These results showed that dietary α-linolenic, eicosapentaenoic and docosahexaenoic acids differently influenced hepatic lipogenesis and the partition of fatty acid into oxidation or glycerolipid synthesis.
We examined the antiperoxidative properties of a fermented bovine milk whey preparation in rats fed on a low vitamin E-containing diet and identified the active principle in the preparation. An exogenous supply of either lactic acid or an amino acid mixture simulated the unfermented whey proteins to prevent red blood cell (RBC) hemolysis and to lower liver thiobarbituric acid reactive substances (TBARS). The supply of either whey proteins or β-lactoglobulin resulted in an increase in liver GSH and prevented iron-mediated lipoprotein peroxidation. These protein effects were reproduced in rats orally administered with either GSH or its precursor, γ-glutamylcysteine. The amount of TBARS formed during in vitro lipoprotein peroxidation were positively correlated with liver TBARS. These results suggest that fermented milk products containing lactic acid and bovine milk whey proteins can ameliorate peroxidative stress in tissues subjected to vitamin E deficiency.
Two potent antioxidative isoflavones were isolated from soybeans fermented with Aspergillus saitoi by silica gel column chromatography and preparative HPLC, using ODS column, or in addition, Toyopearl HW-40 column chromatography. The purified AS-13 and AS-9B compounds were identified as 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), respectively, by MS, and 1H-NMR, 13C-NMR and HMBC spectra. These isoflavones, which have an o-dihydroxy structure between the 7- and 8-position, each exhibited significantly stronger antioxidative activity than daidzein and genistein in both oil and lipid/aqueous systems. Furthermore, the antioxidative activity and the content of each isoflavone analog in soybeans with different fermentation periods were investigated. It is suggested from these results that AS-13 and AS-9B were produced from daidzein and genistein, respectively, by hydroxylation at the 8-position of each isoflavone structure. In addition, it is concluded that these isoflavones were also the principal antioxidants in potent antioxidative soybeans fermented with A. saitoi.
We have previously reported that feeding rats with a diet containing 0.02% L-Nωnitroarginine (L-NNA), a specific inhibitor of nitric oxide synthase, induced hypercholesterolemia. This present study was conducted to examine the underlying mechanism for hypercholesterolemia in rats. In experiment 1, feeding a diet containing 0.02% L-NNA for 5 wk elevated the concentration of serum cholesterol and reduced the excretion of fecal bile acids, but did not affect the excretion of fecal neutral sterols. Reduced activity of hepatic cholesterol 7 α-hydroxylase, the rate-limiting enzyme for the biosynthesis of bile acids from cholesterol, was observed in the rats receiving L-NNA. In experiment 2, rats were fed for 5 wk on a diet with or without 0.02% L-NNA that was or was not supplemented with 4% L-arginine. The L-NNA treatment elevated the serum concentrations of total cholesterol, free cholesterol and esterified cholesterol, and reduced the activity of hepatic cholesterol 7 α-hydroxylase, serum nitrate (a metabolite of NO) and the ratio of HDL-cholesterol versus serum total cholesterol. These alterations were suppressed by supplementing the L-NNA-containing diet with L-arginine. The results suggest that lower NO production by L-NNA caused hypercholesterolemia by a mechanism involving impaired bile acid synthesis.
The autoxidation mechanism for L-ascorbic acid (ASA)-related compounds such as D-arabo-ascorbic acid (=erythorbic acid; ERA) and triose reductone (TR) in methanol without metal ion catalysis was studied. The oxidation reaction of these ASA-related compounds seems to proceed via the C(2) oxygen adduct of ERA (or TR) by a similar reaction mechanism to that of ASA.
Following the administration of theanine, the brain tryptophan content significantly increased or tended to increase, but the contents of serotonin and 5-hydroxyindole acetic acid (5HIAA) decreased. The use of inhibitors of serotonin metabolism enable us to speculate that theanine reduced serotonin synthesis and also increased serotonin degradation in the brain.
Inulin fructotransferase (DFA III-producing) [EC 184.108.40.206] secreted from Bacillus sp. snu-7 was purified 60.3-fold with a yield of 11.6% from a culture supernatant by ammonium sulfate precipitation, preparative isoelectrofocusing, anion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 62 kDa on SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was found to be Ala-Asp-Gly-Gln-Asp-Gly-Ala-Pro-Leu-Asn-Gln-Val-Asn-Thr-Tyr-Asp. The optimal pH and temperature for the enzyme reaction were 6.0 and 40°C, respectively. The enzyme was stable with a pH range of 4.0 to 7.0 and at up to 60°C. As the production of di-D-fructose 1,2′:2,3′ dianhydride increased in the course of enzyme reaction, the Km of the purified enzyme was estimated to be 5.4 mM. One mM each of Cu2+, Fe2+ and Hg2+ inhibited the enzyme activity strongly. Exhaustive enzymatic digestion of inulin produced 1-kestose, 1-nystose, and 1-F-fructofuranosylnystose as well as di-D-fructose 1,2′:2,3′ dianhydride.
We found that a psychrophilic bacterium, Flavobacterium sp., characterized in this study, has a β-mannanase (EC 220.127.116.11) activity in the culture medium. The mannanase activity was the highest in the culture medium, containing 1.0% (w/v) guar gum (as a carbon source), 0.3% (NH4)2SO4 (as a nitrogen source), and 0.06% (w/v) yeast extract, of five-days cultivation at 4°C. No mannanase activity was found in the medium containing a monosaccharide or a disaccharide as a carbon source, although the psychrophile could use them. The enzyme activity was found only when the bacterium was cultured in the medium containing a polysaccharide. The enzyme preparation showed a single activity band on a washed gel of SDS-PAGE. The optimal temperature for the enzyme activity was 35°C. When the reaction was done at 10°C, the enzyme showed 25% of the optimal activity. The β-mannanase preparation efficiently hydrolyzed guar gum, locust bean gum, and glucomannan as well as β-mannan.
The chemical composition of the glue substance that attaches the appressoria of Magnaporthe grisea bar artificial leaf wax was investigated. As a percentage of fresh weights, the glue was crude lipids 29.0%, proteins 12.2%, sugars 7.6%, water 26.5% and other substances 24.7%. The major fatty acid components of the crude lipids were hexadecanoic and octadecanoic acids. Unsaturated fatty acids and branched fatty acids were also detected as minor components. The protein was abundant in glycine, glutamic acid, and serine. Glucose, xylose, and mannose were the major monosaccharides.
A moderately thermophilic bacterium, strain TI-1, produces H2S outside of the cells when grown at 45°C on Fe2+-medium (pH 1.8) containing elemental sulfur and L-glutamic acid. A newly identified sulfur reductase was present in the cytosol of this strain and was purified to an electrophoretically homogeneous state from strain TI-1. The apparent molecular weight of sulfur reductase was 86,000 by gel filtration and 48,000 by SDS-PAGE, so the enzyme was a homodimer. The enzyme was most active at pH 9.0 and 60 to 70°C, and it catalyzed the reduction of 1 mol of elemental sulfur with 1 mol of NADH to give 1 mol of H2S and 1 mol of NAD+. Elemental sulfur was a specific electron acceptor of this enzyme. Thiosulfate, sulfite, and tetrathionate were not electron acceptors, but inhibited sulfur reductase activity. NADPH was not used as an electron donor.
We tried a method for the production of trehalose 6-phosphate (T6P) with energy-coupling fermentation by baker’s yeast. T6P was produced in a reaction mixture containing glucose, 5′-UMP, MgSO4, inorganic phosphate, and dried cells of baker’s yeast as the enzyme preparation. T6P was isolated from the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic methods. The reaction conditions suitable for T6P production were investigated. The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose, at various pHs and concentrations of substrates was examined. Accumulation of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM 5′-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100 mg/ml dried cells of baker’s yeast shaken at 37°C for 6 h. The yield of T6P as a percentage of glucose was 11% (mol/mol) under these reaction conditions.
A neutralo-sensitive mutant (M-12) was isolated from the facultative alkaliphilic Bacillus sp. C-125. This mutant strain was able to grow to the same extent as did the parent strain above pH 8, but did not grow below pH 7.5. The same extent of oxygen uptake was shown by the cells of the parent and mutant strains at pH 10.3. On the other hand, the oxygen uptake rate was about one-fifth of that of the parent strain at pH 7. NADH-dependent oxygen uptake by everted vesicles of the mutant was lower than that of the parent strain at pH 7-7.5, while the rate at pH 8-9 was almost identical in both strains. The activity at pH 7 of cytochrome c oxidase of right-side-out membrane vesicles of the mutant strain was lower than that of the parent strain at pH 7, while both samples had almost the same enzymatic activity at pH 8.5. These results suggest that poor respiratory activities of the mutant strain at pH 7 are the reason why this mutant strain was unable to grow at neutral pH.
In Bacillus sp. YUF-4, acetylacetoin synthase was induced by acetoin, while glucose inhibited the induction. The enzyme was purified 111-fold by 6 purification steps, and a further purification followed, by HPLC using a TSK gel, Phenyl-5PW RP. The resulting enzyme gave a single band with a molecular mass of 62 kDa by SDS-PAGE and 220 kDa by gel filtration. Some enzymic characteristics were studied.
Immobilized mycelia were screened for xylooligosaccharide production from xylan, and 20 strains of Aspergillus oryzae were selected. For its high activity and operational stability of xylooligosaccharides formation, immobilized A. oryzae I3 was selected for further examination. Batch production of xylooligosaccharides from xylan by the immobilized mycelia was repeated a total of 4 times.