5,5′-Diferulic acid (5,5′-DFA), which is one of the cross-linking residues in plant cell walls, was prepared by using a facile procedure. The phenol oxidation of vanillin with FeCl3 gave divanillin, which was further devoted to a Perkin reaction to give the desired product. It was found on 13C-NMR that the chemical shift of C-5 of ferulic acid (FA) clearly shifted downfield, when this carbon is quaternarized by the oxidative dimerizaton to 5,5′-DFA, while those of other carbons of 5,5′-DFA are fundamentally same as those of FA. Also prepared was [9,9′-13C2]-5,5′-DFA, which was proved to be a good internal standard on GC-MS quantification of endogenous 5,5′-DFA from plant tissues.
The root growth-promoting factor, capillarol, and derivatives were synthesized from p-hydroxybenzaldehyde by photo-Fries rearrangement and their root growth-promoting activity for rice seedlings was studied.
Environmental pollution has resulted in the use of organo-tin and organo-copper compounds as antifouling agents being prohibited. Non-toxic natural products are considered to be environmentally friendly antifouling agents. Several phloroglucinol compounds isolated from three species of Eucalyptus have demonstrated strong attachment-inhibiting activity against the blue mussel. In this paper, we discuss the structure-activity relationship of these phloroglucinol compounds on the basis of molecular mechanics calculations. When these compounds were superimposed on each other and the strongest inhibitor was applied as a template, the repulsion energy of the other compounds was proportional to the logarithmic attachment-inhibiting activity. It is concluded that, of the two phloroglucinol rings in sideroxylonal A, ring C is more important, and those compounds that stereochemically and electrostatically resemble the template molecule are more active.
Optically active α-methylbenzylphenylureas were tested for their relieving activities toward just-germinated rice seedlings injured by bensulfuron-methyl (methyl 2- [[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]- sulfonyl]methyl]benzoate) in an agar test to evaluate the chiral requirement and enantioselectivity. Many kinds of derivatives of the α-methylbenzylphenylureas exhibited strong relieving activity without any affect on root growth at the highest concentration tested. Six compounds with an (S)-configuration were more active than daimuron. The log k′ values of the most potent derivatives ranged from 0.42 to 0.65. A relatively strong parabolic relationship between the log k′ value and the activity has only been found in the case of the (S)-enantiomers containing halogen atoms. The enantioselectivity of the chiral pairs was very high, and the chirality-activity function followed a Pfeiffer relationship, increasing the selectivity with increasing potency. Among them, the 2,3-Cl2, 2-F-4-CH3, 4-COOEt, 2-Cl and 2,5-F2 derivatives were highly enantioselective with a significantly high relieving activity. These results suggest that while hydrophobicity performed an important role, chirality and the mode of substitution essentially contributed to the activity.
To test the stereogenic effect of jasmonate on phytohormonal activities, methyl 7-epi-jasmonate (1b) and four stereoisomers of methyl cucurbate were easily prepared in racemic form: epimerization at the C-7 position of a commercially available methyl jasmonate (2b) with a base and subsequent fractional distillation gave a 46:54 mixture of 1b and 2b, whose reduction gave a mixture of methyl cucurbates (3-6). This synthetic chemistry was supplemented by molecular modeling and an NMR study on 1b and 2b. An assessment of the inhibitory activities of the prepared jasmonates on growth of the second leaf sheath of rice and on seed germination of cress clarified that the cis-configuration of the C-3 and C-7 side chains of jasmonate was an important factor for the high activities. In inhibiting the seed germination of cress, methyl 6-epi-cucurbate (4) exhibited activity that was markedly higher than the other compounds tested, showing that the stereochemistry at C-6 as well as at C-3 and C-7 was strictly recognized by this bioassay.
The indophenol-reducing compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (I), was isolated from the culture filtrate of an unidentified fungus. I also reacted with the DPPH radical to form a reaction product IV which was determined to be 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo- 2H-pyran-3-yl)phenyl]-1-phenyl-2-picrylhydrazine. This is the first report describing the formation of an adduct of the DPPH radical and its scavenger.
The mixed anhydride method was applied to synthesize O-aryl O-ethyl phenylphosphonothionate. The reaction of O, O-diethyl phosphorochloridate with O-ethyl phenylphosphonothioic acid afforded O, O-diethyl phosphoric O-ethyl phenylphosphonothioic anhydride in a good yield. This anhydride was converted to O-aryl O-ethyl phenylphosphonothionates by reacting with the appropriate sodium phenoxide. This esterification occurred without racemization and produced optically pure O-ethyl O-(4-nitrophenyl) phenylphosphonothionate (EPN) and O-(4-cyanophenyl) O-ethyl phenylphosphonothionate (cyanofenphos).
A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.
A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.
Vibrio alginolyticus, a halophilic marine bacterium, produced an extracellular amylase with a molecular mass of approximately 56,000, and the amylase appeared to be subject to catabolite repression mediated by cAMP. The production of amylase at pH 6.5, at which the respiratory chain-linked H+ pump functions, was inhibited about 75% at 24 hours following the addition of 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), while the production at pH 8.5, at which the respiratory chain-linked Na+ pump functions, was only slightly inhibited by the addition of 2 μM CCCP. In contrast, the production of amylase in a mutant bacterium defective in the Na+ pump was almost completely inhibited even at pH 8.5 as well as pH 6.5 by the addition of 2 μM CCCP.
We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.
The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region.
We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier.
We have searched for phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3) binding proteins in Dictyostelium discoideum using beads bearing a PIP3 analogue, PIP3-APB. One of the binding proteins with a molecular mass of 55 kDa was purified and its amino acid sequence was partially amalyzed. Database searches showed that the analyzed sequence was identical to that of protein kinase B (PKB) of D. discoideum. The specific activity of D. discoideum PKB, when expressed together with constitutively active PI-3 kinase in mammalian cells, was elevated by about three-fold, suggesting that PKB could also act downstream of PI-3 kinase in Dictyostelium cells.
We examined the relationship between cell proliferation and differentiation of cultured rat fetal and newborn hepatocytes isolated from various developmental stages. The albumin production rate increased along with cell growth under in vitro culture and became maximal two days after the growth cessation. AFP was secreted by both fetal and newborn hepatocytes with growth ability. Furthermore, the responses to HGF addition in fetal hepatocyte cultures were observed in terms of growth stimulation and down-regulation of the Met receptor. We also studied the changes in RB and liver enriched transcription factors (C/EBPs) for investigating the mechanism underlying proliferation and differentiation of fetal hepatocytes. Western blot analysis of hepatocytes taken from various gestation stages of rat liver showed that the expression of RB and C/EBPβ increased as gestation stage proceeded. When RB antisense S-oligonucleotide was added to the culture medium, proliferation and AFP expression increased, while C/EBPα and albumin expressions decreased. These results indicated that the tumor suppressor gene product RB had a profound role not only in cell proliferation but also hepatocyte differentiation.
The Escherichia coli EnvZ-OmpR regulatory system is a paradigm of intracellular signal transduction mediated by the well-documented phosphotransfer mechanism, by which the expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. Although it is clear that the EnvZ histidine(His)-kinase is the major player in the phosphorylation of OmpR, it has been assumed for some time that there may be an alternative phospho-donor(s) that can phosphorylate OmpR under certain in vitro and in vivo conditions. In this study, to address this long-standing issue, extensive genetic studies were done with certain mutant alleles, including ΔenvZ, Δ(ackA-pta), and ΔsixA, as well as ΔompR. Here, for the first time, genetic evidence is provided that, in addition to EnvZ, acetyl phosphate and an as yet unidentified sensor His-kinase can serve as alternative in vivo phospho-donors for OmpR, even in the envZ+ background. A model for the alternative phosphotransfer signaling pathway involved in the phosphorylation of OmpR is proposed.
A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.
Possible differences between two representative non two-state thermal unfolding mechanisms of protein are discussed concerning differential scanning calorimetry. Numerical simulations showed that, by DSC measurement, it is hard to discriminate between the independent model, which assumes independent unfolding domains in a protein, and the sequential model, which assumes intermediate(s) between native and denatured states, especially when values of molecular weight, denaturation enthalpy, and difference in denaturation temperature of each denaturation process are large. DSC curve analysis of Aspergillus niger glucoamylase based on these two models gave essentially the same thermodynamic parameters.
Earthworm and leech cDNAs encoding the GGNG peptides, a family of myotropic peptides, were cloned and examined in this study. Both of the predicted precursor proteins are of polyprotein structure and contain several putative peptides distinct from the GGNG peptides. However, the precursors show organizations distinct from each other and no sequence similarity except for the GGNG peptides.
Aqualysin I is the alkaline serine protease isolated from an extreme thermophile, Thermus aquaticus YT-1. We have analyzed the kinetic properties of aqualysin I, using thirty-one kinds of chromogenic succinyl-tripeptide p-nitroanilides as substrates in the presence of 10% dimethylsulfoxide (DMSO). Aqualysin I hydrolyzed many peptides in a DMSO-containing mixture, however the substrate specificity was different from that in the absence of DMSO. The Km for each peptide was raised by the addition of 10% DMSO. Also, the P3- as well as P2-specificity of aqualysin I was altered. These results suggested that the side chains of the P2 and P3 residues are exposed to the solvent, and the hydrophobic interactions between the side chain of the substrate and the solvent may take a part in the substrate recognition of the enzyme.
Novel dolichyl derivatives were found in rat spleen. The compounds were eluted from reverse phase HPLC after eluting dolichyl fatty acid ester. The elution profiles of the unsaponified forms of the unknown compounds were coincident with that of dolichol from spleen on reverse phase HPLC. The compounds were not dolichyl dolichoate, which are present in bovine thyroid. The compounds were not found in young rats (4 months of age) but were found in old rats (above 12 months of age), and they were not detected in other tissues under our conditions.
Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.
The water sorption and drying behavior of crosslinked dextran gels was investigated in relation to the dependency on the density of the crosslinks. Sorption isotherms indicate that the amount of sorption was more for the gels with a lower crosslink density. Almost all the isotherms show sorption and desorption hysteresis, the extent of this hysteresis being more marked with the gels with lower crosslink density. The rate of drying was also dependent on the crosslink density. These results can be explained by considering that the polymer network in a gel with a low crosslink density is more flexible to easy change during water sorption and drying than in one with a higher crosslink density.
The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5′-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 μM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10-4 M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.
Apolipoprotein (apo) E-deficient mice were fed an atherogenic diet with either 1% ethyl ester docosahexaenoic acid (DHA) or safflower oil (SO) as a source of linoleic acid for 8 week. Both genders fed DHA had higher proportions of eicosapentaenoic acid and DHA, and lower proportions of linoleic and arachidonic acids in the liver and serum phospholipids than those fed SO. Males fed DHA had greater liver weight and tended to have higher concentrations of serum lipids and liver cholesterol than those fed SO, and there were opposite trends in females. Dietary fats and gender led to no significant effect on lesion sizes in aortic arch and thoracic plus abdominal aorta. These results indicate that the interactive action of sex-related factor(s) with dietary polyunsaturated fatty acids is involved in metabolic changes of serum lipids in apoE-deficient mice, and addition of DHA, compared with addition of SO, is not effective to abolish the atherosclerosis in this animal model.
The composition of nutritionally and physiologically important molecules in transgenic rice with the soybean glycinin gene was determined and compared with that of a non-transgenic control. Except for the levels of protein, amino acids and moisture, no marked differences were found between the two kinds of rice. The protein content of the transgenic rice was about 20% higher than the control (control, 6.5 g/100 g; transgenic, 8.0 g/100 g) with a concomitantly lower moisture content. This increased protein content mainly resulted from the increased glycinin expressed in the transgenic rice, and the protein was susceptible to gastric and intestinal digestion juices. In parallel with the increased protein content, some important amino acids lacking in quantity in normal rice were replenished.
The protective effects of various kinds of dietary amino acids against the hepatotoxic action of D-galactosamine (GalN) were examined. Male Wistar rats fed with 20% casein diets containing 10% or 5% amino acid for one week were injected with GalN (800 mg/kg body weight), and the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, the hepatic glycogen concentration, and the serum glucose-level were examined 20 hours after the injection. In the groups with the 10% amino acid diets, activities of AST, ALT, and LDH in serum of 10% L-glutamine (Gln), 10% L-asparagine (Asn), and 10% L-serine (Ser) groups were significantly lower than those of the control group, and in the groups with the 5% amino acid diets, those activities of 5% L-histidine (His), 5% L-tyrosine (Tyr), 5% L-lysine (Lys), and 5% L-glycine (Gly) groups were also lower than those of the control group. The concentration of liver glycogen of 10% Gln-, 10% Asn-, and 10% Ser-groups and those levels of 5% His-, 5% Tyr-, 5% Lys-, and 5% Gly-groups were also significantly higher than that of the control group. As a result, it was found that some kinds of dietary amino acid such as L-Ser, L-Asn, L-His, L-Lys, L-Tyr, and L-Gly, in addition to L-Gln were effective to protect the rats from GalN-induced injury.
The protective effect of flavonoids against linoleic acid hydroperoxide (LOOH)-induced cytotoxicity was examined by using cultured endothelial cells. When the cells were incubated with both LOOH and flavonoids, most flavonols protected the cells from injury by LOOH. Flavones bearing an ortho-dihydroxy structure also showed a protective effect against the cytotoxicity of LOOH. However, flavanones had no effect. The structure-activity relationship revealed the presence of either the ortho-dihydroxy structure in the B ring of the flavonoids or 3-hydroxyl and 4-oxo groups in the C ring to be important for the protective activities. The interaction between flavonoids and a-tocopherol was also examined in this system. Flavonoids that were protective against LOOH-induced cytotoxicity had at least an additive effect on the action of α-tocopherol against LOOH-induced damage.
The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.
The immunopotentiating activity of nigerooligosaccharides (NOS), a mixture of nigerose, nigerosyl glucose and nigerosyl maltose, was studied in vitro and in vivo in mice. Mitogen-induced proliferation of splenocytes from normal mice was augmented in a dose-dependent manner by nigerose of NOS. NOS enhanced interleukin 12 (IL-12) and interferon-γ (IFN-γ) production by normal splenocytes in the presence of the potent IL-12 inducer, heat-killed Lactobacillus plantarum L-137, in vitro. Consistent with the in vitro finding, L. plantarum L-137-induced IL-12 production and IL-2-induced IFN-γ production were augmented in mice fed with a 14.6% NOS diet for 2 weeks compared with mice fed with a control diet. Notably, mice fed with the NOS diet showed significantly longer survival time than the control mice after the induction of an endogenous infection by administering 5-fluorouracil in a lethal dose. Taken together, these results suggest that NOS may exert immunopotentiating activity through the activation of an IL-12-dependent T helper 1-like immune response.
A study was carried out to establish an animal model that would be suitable for evaluating the role of the diet in immune cell-mediated atherogenesis. Brown Norway rats were initially treated with hypervitamin D2 for 4 days and then fed on an atherogenic diet for 3 months, during which period the rats were either immunized with ovalubumin plus Al(OH)3 (OVA group) or with Al(OH)3 alone (control group) every 3 weeks. Aortic lesions were mainly composed of foam cells, the lesions evaluated by the intimal thickness of the ascending aorta being more severe in the OVA group than in the control group. The OVA group, in comparison with the control group, showed prominently increased serum levels of OVA-specific IgG and rat chymase, an indicator of mast cell degranulation. The intimal thickness was positively correlated with the level of chymase. Immunization had no effect on the serum lipid levels. These results support the hypothesis that mast cells play a role in the early stage of atherosclerosis and suggest that this animal model could be useful for evaluating the role of the diet in immune-related atherogenesis.
A glycosidically bound fraction was prepared by adsorbing a 80% methanol extract from fresh rhizomes of ginger onto a column of Amberlite XAD-2 resin and successively eluting with ethyl acetate or methanol. Enzymatic hydrolysis of this fraction with an acetone powder prepared from fresh ginger and commercial glycosidase liberated such alcohols as geraniol, 2-heptanol, α-terpineol, nerol, linalool, and citronellol, suggesting that fresh ginger included glycosides and glycosidase. The ethyl acetate eluate was chromatographed by an ODS flash column and then HPLC to isolate the β-glucopyranosides of 5-hydroxyborneol, 1,8-epoxy-p-menthan-3-ol, (2E, 6E)- and (2E, 6Z)-3,7-dimethyl-8-hydroxyoctadien-1-ol, 2-heptanol, geraniol, nerol, (R)-linalool, and citronellol. All the glucosides, except for 5-hydroxybornyl-O-β-D-glucopyranoside, were identified for the first time in the rhizome of ginger, and many of their aglycons were major constituents of the essential oil. The results indicate that these glucosides are aroma precursors of fresh ginger.
The effectiveness was evaluated of a cathodic solution prepared by the electrolysis of an NaCl solution in inhibiting the aqueous oxidation of ethyl linoleate and ethyl docosahexaenoate. The decrease in unoxidized substrate and the formation of total peroxides during oxidation indicate that the cathodic solution completely inhibited the oxidation of both ethyl esters, while these lipids were easily oxidized in an NaCl solution and in distilled water. The antioxidative activity of the cathodic solution was confirmed after open incubation for 3 days and 7 days at 37°C, although the scavenging ability of the cathodic solution toward DPPH radicals disappeared during this incubation.
Methylation of (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) was carried out with a rat liver homogenate and S-adenosyl-L-methionine. A structural analysis of the reaction products by MS and NMR showed that 4′-O-methyl EGC, 4″-O-methyl ECg, and 4″-O-methyl EGCg had been formed from EGC, ECg, and EGCg, respectively. These results suggest that methylation may be one of the metabolic pathways to the catechins.
An extracellular β-glucosidase was purified from a culture filtrate of the fungus Cladosporium resinae strain NK-1 grown on a medium containing starch, Tween 80, and yeast extract. The purified enzyme was monomeric with an Mr 98,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration using HPLC. The enzyme had optimal activity with p-nitrophenyl-β-D-glucoside (PNPG) at 50°C and pH 4.5. The enzyme catalyzed the hydrolysis of cellobiose and PNPG. The Km and Vmax with PNPG as the substrate at 50°C and pH 4.5 were 0.07 mM and 364 μmol/min/mg, respectively; with cellobiose as the substrate, the corresponding values were 2.3 mM and 75 μmol/min/mg. The enzyme activity was competitively inhibited by glucose (Ki=20 mM), while fructose, galactose, mannose, arabinose, xylose (each at 50 mM), sucrose, and lactose (each at 30 mM) were not inhibitory. While the enzyme has activity against sophorose (β-1,2-glucobiose) and laminaribiose (β-1,3-glucobiose), it has no activity against gentiobiose (β-1,6-glucobiose). The activity of the β-glucosidase was inhibited by Ag+, Fe2+, Mn2+, Zn2+, Hg2+, SDS, and p-chloromercuribenzoate.
A stress imposed by a continuous feed of high ethanol, high NaCl concentration, or a high temperature shock increased antibiotic production by several times in Pseudomonas fluorescens S272. A tentative bioassay showed that the stress caused about 40-fold elevation in the autoinducer activity. Addition of synthetic autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone or N-(3-oxohexanoyl)-L-homoserinelactone at a concentration of more than 100 μg/l to a non-stressed culture also increased the antibiotic production by several times. These results suggested that the antibiotic production in P. fluorescens S272 was regulated by N-acyl-homoserine lactone and the promotive effect by stress occured through any function that increased the autoinducer production.
Microorganisms that accumulated the eicosapentaenoic acid (EPA)-enriched triacylglycerol (TG), were screened for using yeast-malt medium containing 1% free EPA. The best strain was identified as Mucor hiemalis HA-30. The optimum culture conditions for the accumulation of EPA-enriched TG were : 3% soluble starch, 0.5% polypeptone, 0.3% yeast extract, 0.5% free EPA, and pH 6.0 at 25°C. After the cultivation, 1.77 mg/ml of the TG with EPA purity of 79% was obtained. The EPA content in TG increased in conjunction with the EPA content of the supplemented free fatty acids or ethyl esters. Free EPA were more efficiently incorporated than the ethyl esters. Trieicosapentaenoyl glycerol (EPA, EPA, EPA) accounted for 73% of total TGs.
D-Mannitol dehydrogenase (EC 22.214.171.124) was purified and crystallized for the first time from the cell-free extract of Gluconobacter suboxydans IFO 12528. The enzyme was purified about 100-fold by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration by a Sephadex G-75 column. The enzyme was completely separated from a similar enzyme, NAD-dependent D-mannitol dehydrogenase (EC 126.96.36.199), during enzyme purification. There being sufficient purity of the enzyme at this stage, the enzyme was crystallized, by the addition of ammonium sulfate, to fine needles. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.6 s. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE and gel filtration chromatography. Oxidation of D-mannitol to D-fructose and reduction of D-fructose to D-mannitol were specifically catalyzed with NADP and NADPH, respectively. NAD and NADH were inert for the enzyme. Since the reaction equilibrium declined to D-fructose reduction over a wide pH range, the enzyme showed several advantages for direct enzymatic measurement of D-fructose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the D-fructose reduced.
We attempted to breed phage-insensitive strains of Tetragenococcus halophila D10. Phage contact during selection initially caused the occurrence of lysogeny. Subsequently, we screened phage-insensitive mutants by replica plating so that mutant cells did not touch the phage during selection. Two strains were selected from about 150,000 strains. They grew normally in soy sauce mash (moromi) in the presence of phage φD-10, although they had a similar extent of adsorption of φD-10 as did the parent strain.
The cold-sensitive fcsA29 mutation of Escherichia coli was found to be a new type of cold-sensitive allele of the polA gene encoding DNA polymerase I, caused by an Asp(116)→Asn change in the 5′→3′ exonuclease domain. The fcsA29 mutant showed typical polA mutant phenotypes such as UV sensitivity and unacceptability of recA mutation. Cold-sensitive growth of the mutant was suppressed by introduction of a sulA mutation, indicating that cell filamentation was due to the SOS response.