W493 A and B, which showed strong antifungal activity, were isolated from a culture broth of Fusarium sp. The structure of W493 B was determined to be that of a cyclodepsipeptide, cyclo(3S,4R-HMTA-D-allo-Thr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Ile) (1) by MS and NMR data, an amino acid analysis, and synthesis of the component. HMTA represents 3-hydroxy-4-methyltetradecanoic acid. W493 A (2) had a similar structure, except that L-Ile in 1 was replaced by L-Val. The absolute configuration of each amino acid was determined by chiral HPLC, and the sequence of the components was determined by HMBC experiments. The sequence of the two alanines was determined to be a L-Ala-D-Ala by a chiral HPLC analysis of the peptide fragment containing only one Ala residue. The absolute configuration of HMTA obtained from the hydrolysis of W493 B was determined to be 3S and 4R by comparing with four isomers prepared by enantioselective synthesis via Sharpless asymmetric epoxidation.
Aspergillus oryzae O-1018 (FERM P-15834) separated from industrial koji for brewing sake was found to produce five papain-inhibitory compounds in the culture supernatant. The five isolated inhibitors were named CPI-1 to CPI-5, and their structures were elucidated by spectroscopic analyses and chemical degradation. We determined the structures of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N- [(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl] amino]butane, 5-amino-1-[[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]amino]pentane and N8- [N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu-cyl]spermidine, respectively. We also confirmed by a degradation experiment that CPI-1 consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine and spermidine. Although CPI-4 was identified as kojistatin A,1) the other CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition potency of CPIs against cysteine proteases was as high as or higher than that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times more inhibitory than E-64 toward cathepsin L.
Manauealide C (1) and anhydrodebromoaplysiatoxin (4), toxic constituents of the Hawaiian red alga, Gracilaria coronopifolia which has been concerned with food poisoning cases, were studied. The absolute structure of manauealide C was determined as 1 by chemical conversion and spectroscopic methods. The first complete assignment of 13C chemical shifts for anhydrodebromoaplysiatoxin (4) was established. The biological activity of 4 was also investigated.
Three endopolygalacturonases (endoPG Ia, Ib, and Ic) were isolated from the culture filtrate of Stereum purpureum, the causative fungus of apple silver-leaf disease. Their properties, including specific activities, optimum pHs, thermal stabilities, and kinetic parameters (Km and Vmax) were compared. Their properties were very similar to one another except for the substrate specificity and relative molecular mass. The sugar chains of endoPG Is were released by hydrazinolysis, and one major sugar chain common to endoPG Is was isolated. The pyridylamino sugar was characterized by a two-dimensional mapping method using HPLC, and identified as a high mannose type N-linked sugar chain, Manα1-6(Manα1-3)Manα1-6(Manα1-3) Manβ1-4GlcNAcβ1-4 GlcNAc (designated as M5.1). Observation of the course of Western blot analysis for the proteins from the culture filtrate with endoPG I antibodies showed that the fungus secreted three endoPG Is into the culture broth during the growing period.
Two azurin-type blue copper proteins, which is concerned with the electron transport chain involved in methylamine/methanol oxidation, have been found in the obligate methylotroph Methylomonas sp. strain J. The azurin iso-1 gene was cloned and sequenced to analyze the role in the electron transport chain. PCR products synthesized with primers based on the N- and C-terminal amino acid sequences of azurin iso-1 were used as probes for cloning. One complete open reading frame (the azurin iso-1 gene) and one partial orf (orf1) were found in a cloned Eco105I-HindIII fragment, pMAZ3, with a total of 1066 bp. The gene encoded 148 amino acid residues. The amino acid sequence after Ala-21, deduced from the nucleotide sequence, was identical to that of the azurin iso-1 protein. The gene was in a region separate from the mau gene cluster in the chromosome. Escherichia coli expressed azurin iso-1. The results of northern blotting analysis suggested that expression of the azurin iso-1 gene is regulated by a complex regulatory network controlling oxidation of methylamine or methanol in this strain; for example, copper ions affected the expression of the azurin iso-1 gene.
Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65°C, and the enzyme was stable at 55°C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.
For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45°C with N-carbamyl-D-amino acids as the sole nitrogen source. The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction. One of the enzymes, produced by Pseudomonas sp. strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced. A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli. The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000. The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp. strain KNK712, was 60%. A database was searched for similar sequences.
The amino-carbonyl reaction (Maillard reaction), also known as glycation, of egg-yolk phosphatidylethanolamine (PE) was induced by incubating PE (50 mg/ml) with D-glucose (222 mM) in a methanol medium containing 2,6-di-tert-butyl-p-cresol as an antioxidant at 37°C for 4 days. The resultant product, glycated PE (gPE), was then isolated from the reaction mixture by two-step normal and reversed-phase high-performance liquid chromatography with UV diode array detection and was characterized as having a 1:1:1 elemental ratio of phosphorus:nitrogen:sugar. The Fourier transform-nuclear magnetic resonance spectrum and infrared absorbance spectrum indicate the isolated gPE to have been deoxy-D-fructosyl PE, which is an Amadori product of PE. The fast atom bombardment-mass spectrometric data for the glycation product of authentic dioleoyl PE (1,2-di-9-octadecenoyl-sn-glycero-3-phosphoethanolamine) show that the molecular weight of gPE corresponds to that of glucose-conjugated PE in the form of an Amadori product. This Amadori product formation was also confirmed in PE/phosphate buffer dispersions and in phosphatidylcholine-PE liposome/phosphate buffer suspensions in the presence of D-glucose at 37°C. gPE was degraded by phospholipase A2, C and D. Freshly spiked blood plasma and red blood cells (RBC) from normal human volunteers contained substantial levels of gPE, the concentration corresponding to at least 9 mol% of PE. Remarkable formation of gPE, up to 15-45 mol% of PE in human blood plasma and RBC, was further confirmed by prolonged incubation with 5-45 mM D-glucose. The gPE formation in RBC was found to be proportional to the glycated hemoglobin formation.
In several organisms that form fruit bodies, the synthesis of lectins is developmentally regulated. Aleuria aurantia is an ascomycete that forms a fruit body known as orange peel mushroom. To find whether the mycelia of this organism synthesize a lectin, mycelial isolates were obtained from a wild fruit body by spore germination and regeneration from a fragment of the fruit body. The isolates were identified as A. aurantia by analysis of their DNA. The mycelial isolates synthesized a lectin with the same properties as those of fruit-body lectin in terms of subunit molecular mass, immunochemical reactivity, binding specificity for L-fucose, and N-terminal amino acid sequence. Vegetatively growing mycelia synthesized as much lectin as the fruit body, so such synthesis was not developmentally regulated, unlike some other organisms that form fruit bodies.
We investigated the effects of free radical generation on the esterification of cholesterol by lecithin-cholesterol acyltransferase (LCAT). A water-soluble free radical initiator, 2,2′-azobis-amidinopropane dihydrochloride (AAPH), inhibited the activity of plasma LCAT as a function of the incubation time after its addition. When a small amount of oxidized HDL was added to plasma, LCAT activity was dose-dependently inhibited. To identify the effects of HDL oxidation on LCAT activity, a purified enzyme and cofactor in a vesicle solution (an artificial substrate) were used. i) LCAT activity was inhibited by the oxidation of substrate vesicles, this inhibition being related to the degree of oxidation. ii) This inhibition was observed even if apolipoprotein A-I was not oxidized. iii) Oxidized phosphatidylcholine, but not oxidized cholesterol, in the vesicles affected LCAT activity. iv) The addition of 0-40% of oxidized vesicles to normal substrate vesicles resulted in the activity of LCAT being inhibited in a dose-dependent manner. These results suggest that the esterification of cholesterol by LCAT may be affected by the oxidation of substrate phosphatidylcholine via free radical generation in the plasma.
Polyclonal antibodies against cysteine synthase (CSase; EC 18.104.22.168) isozymes 1, 2, and 3 were used for the detection of complexes of these isozymes with serine acetyltransferase (SATase; EC 22.214.171.124). SATase was partially purified and found to complex with these isozymes by western blotting and immunotitration. When the complexes were treated with a high concentration of O-acetyl-L-serine, they did not dissociate. However, some complexes with CSase 1 or 3 dissociated when left for 24 h at 4°C. Results of western blotting on SDS-PAGE showed that CSase 2 complexed with SATase. CSases 1, 2, and 3 all could complex with SATase, but the tightness of the bond differed.
Effects of chemical modifications on the protein-synthesis inhibitory (PSI) activities of momordin-a and luffin-a were investigated. Treatment with a 50-fold excess of diethylpyrocarbonate at pH 6.5 modified one histidine residue in momordin-a and luffin-a and reduced their PSI activities to 10% and 8.3%, respectively. Modifications with a 20-fold excess of KI3 at pH 7.0 at 0°C greatly reduced their PSI activities to 10% by iodination of nearly one tyrosine residue. The PSI activity of momordin-a was rapidly reduced to 6.4% by the modification of one lysine residue with trinitrobenzensulfonic acid as in the case of luffin-a reported previously. By analyses of the tryptic peptides from the modified momordin-a and luffin-a, the modified residues were identified as His140, Tyr165, and Lys231. Furthermore, the amounts of three modified momordin-a binding to rat liver ribosomes were reduced to about half or less than half of that of native momordin-a. From these results, it was suggested that His140, Tyr165, and Lys231 are highly exposed on the surface of momordin-a and luffin-a molecules and are involved in their PSI activities, probably by binding to ribosomes.
A bifunctional α-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited α-amylase from germinating rice seeds weakly. It inhibited rice α-amylase more than barley α-amylase, and the inhibition of rice α-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian α-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.
Cytosolic free calcium ion concentration ([Ca2+]cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca2+-sensitive luminescent protein, aequorin, when combined with coelenterazine. SA induced a transient [Ca2+]cyt elevation that was dependent on the concentration of SA and pH of the SA solution. The SA-induced [Ca2+]cyt elevation was not reduced in Ca2+-deficient medium, suggesting that Ca2+ was mobilized from an intracellular Ca2+ store(s). Benzoic acid, butyric acid and sorbic acid did not induced a [Ca2+]cyt elevation.
Two polyclonal antibodies and three monoclonal antibodies specific to the etofenprox insecticide were prepared from rabbits and mice, respectively. The monoclonal antibodies were more reactive with etofenprox than the polyclonal antibodies by C-ELISA. Monoclonal antibody cl 205-65 was found to be the most tolerant to methanol, most highly reactive and most specific to etofenprox among the antibodies tested.
The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions of lymphocyte subsets in BALB/c mice were investigated. The proportion of lymphocytes from the thymus and spleen of mice given no B6, that were CD4+CD8- T cells, was larger than in mice given B6, and the ratio of CD8+ to CD4+ T cells in the thymus of mice given no B6 was lower. The concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro with concanavalin A were significantly higher in the mice with B6 deficiency, as was their plasma corticosterone concentrations. These results suggested that B6 is necessary to maintain cytokine levels and lymphoid function in the thymus and spleen of mice.
Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.
We have previously reported that (−)-epigallocatechin gallate (EGCg) inhibited lung carcinoma cell adhesion to fibronectin (FN) and demonstrated its interaction with FN. In the present work, we studied the interaction between thermolysin fragments of FN and EGCg. An amino acid sequence analysis of the fragment bound by EGCg-agarose provided its identification as a carboxyl-terminal heparin-binding domain. Thus, the inhibition of cancer cell adhesion to FN by EGCg is not caused by its direct binding to the cell-binding domain containing an Arg-Gly-Asp-sequence.
A cDNA encoding the human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate (APS) kinase was cloned and sequenced. The enzyme contains an APS kinase domain in its N-terminal portion and an ATP sulfurylase domain in its C-terminal portion. Recombinant full-length enzyme and its constituent APS kinase and ATP sulfurylase domains were individually expressed, purified, and shown to have their respective enzymatic activities.
Difructose anhydride III (DFA III; di-D-fructo-furanose 1,2′:2,3′ dianhydride) was prepared from inulin with Arthrobacter sp. H65-7 inulin fructotransferase (depolymerizing) (inulase II; EC 126.96.36.199). DFA III is not hydrolyzed by enzymes in the small intestine, but is metabolized by microorganisms in the large intestine. We investigated the effects of DFA III on calcium absorption in two experiments. In the in vivo experiment, we examined the effects of DFA III, fructooligosaccharides, and raffinose on calcium absorption in male Sprague-Dawley rats 5 weeks old at start of the experiment and given feed containing 3% of one of these oligosaccharides for two weeks. The apparent calcium absorption was significantly higher in rats fed any of these oligosaccharides than in control rats, and the increase with DFA III was the greatest. Absorption in both the small and large intestines was affected. In rats fed DFA III, the cecal wall thickened and soluble calcium and the amounts of some organic acids were higher than in the control groups. In an in vitro experiment with everted jejunal and ileal sacs of rats, calcium absorption was higher when DFA III was present in the mucosal fluid at all concentrations tested (up to 200 mM). In the jejunal sacs, the increase in calcium absorption depended on the DFA III concentration. In the ileal sacs, the absorption was maximum at 50 mM DFA III and did not increase further at higher concentrations. These results indicate that intact DFA III stimulates calcium absorption in the small intestine, and that cecal fermentation of DFA III may contribute to the increase in calcium absorption by the large intestine.
This study clarifies the effect of exposure to cigarette smoke on L-ascorbic acid (AsA) metabolism and on the activities of drug-metabolizing enzymes. Male Wistar rats were used. The test rats (group T) were exposed to sidestream smoke from cigarette for 2 h every day for 25 days. During the experimental period, the excreted amount of AsA in the urine from group T was higher than that from the control group (group C). At the end of the experimental period, the AsA content of the plasma and tissues, the liver cytochrome P-450 content and the activities of drug-metabolizing enzymes in group T were each higher than those in group C.
A subjective evaluation of beer drinkability and the degree of stomach fullness were found to correlate with the relaxed cross-sectional area of the pylorus antrum measured by real-time ultrasonography. Five kinds of beer with a different malt/adjuncts ratio and degree of attenuation were used. Each beer was given to 9 healthy volunteers at the rate of 3 ml/kg/15 min, and they each recorded the degree of stomach fullness, desire to drink and tastiness every 30 min. With increasing volume drunk, the degree of tastiness and desire to drink were lowered, and the degree of stomach fullness raised. The relaxed cross-sectional area of the pylorus antrum measured by an ultrasonic image analyzer every 30 min was highly correlated with the degree of stomach fullness, tastiness of the beer, and desire to drink (p=0.0021, <0.0001, 0.001). The beer giving the lowest degree of stomach fullness was appraised to be tasty and highly drinkable. These findings suggest that the rate of gastric emptying is one of the factors determining the drinkability of beer, and that measurement of the relaxed cross-sectional area of the pylorus antrum is useful to evaluate stomach fullness during beer drinking.
Maillard reaction products were prepared by heating xylose and lysine at pH 9.0 and 100°C for 3 h, and then fractionated by ethyl ether and ethanol into acidic, neutral and basic low-molecular-weight, ethanol-soluble and ethanol-insoluble fractions. The ethanol-soluble and -insoluble fractions were the major fractions of the xylose-lysine Maillard reaction products (XL MRPs), contributing 79.5% and 20.1%, respectively. XL MRPs revealed an inhibitory effect on linoleic acid peroxidation induced by the Fenton reaction, but did not inhibit liposome peroxidation induced by Fe2+, where it had a prooxidative action. XL MRPs caused oxidative damage to deoxyribose and 2′-deoxyguanosine (2′-dG) induced by the Fenton reaction. The ethanol-soluble and -insoluble fractions also caused oxidative damages while the low-molecular-weight fractions displayed an antioxidative effect in inhibiting the oxidative damage to deoxyribose that was induced by the Fenton reaction. The prooxidative action of the ethanol-soluble and -insoluble fractions resembled that of the untractionated products in the 2′-dG assay. In these systems with deoxyribose, 2′-dG, linoleic acid and liposomes, XL MRPs exhibited either antioxidative or prooxidative properties, which might have been due to competition between their reducing power and scavenging activity toward the hydroxyl radical. However, the low-molecular-weight fractions did not show any prooxidative activity in these oxidation systems.
Gassericin A, a bacteriocin from Lactobacillus gasseri LA39, was purified to homogeneity from the culturesupernatant mainly by reverse-phase chromatography. The molecular weight of gassericin A was found to be 5,652 by mass analysis, unlike the estimated 3,800 found by SDS-PAGE. However, when the purified preparation was treated with lysylendopeptidase, it migrated as a single band to 5,600 with bacteriocin activity on SDS-PAGE. N- and C-terminal amino acids could not be identified. The internal amino acid could be identified after gassericin A was hydrolyzed with lysylendopeptidase. The DNA of the structural gene of gassericin A was sequenced by cloning of the gene from chromosomal DNA with an oligonucleotide probe. The structural gene of gassericin A was found on the chromosomal DNA as an open reading frame encoding a protein composed of 91 amino acids. The amino acid sequence of mature gassericin A was predicted to be 58 residues from the DNA sequence and results of mass analysis. These results suggested that gassericin A has a closed circular structure with N- and C-terminals linked. Gassericin A is a hydrophobic class II bacteriocin; it was 98% identical with acidocin B produced by Lactobacillus acidophilus M46.
Weakening of the Z-disks of skeletal muscle myofibrils contributes to the tenderization of meat during post-mortem aging. To elucidate the weakening mechanism, we compared Z-disks weakened by post-mortem aging of chicken breast muscle with those of myofibrils treated with a solution containing 0.1 mM CaCl2 and 1 μM calpastatin domain I. In both cases, the Z-disks were weakened with a corresponding liberation of their constituent phospholipids (PLs). The liberation of PLs specific to 0.1 mM calcium ions was minimal at pH 6.5 and maximal at 35°C together with the Z-disk weakening. Binding of calcium ions to PLs in the Z-disks was determined by 45Ca-autoradiography. Acidic PLs were strongly radioactive and neutral PLs were appreciably radioactive. It is very probable that acidic PLs would bind electrostatically to α-actinin under physiological conditions, and that this interaction would be broken by the binding of calcium ions at 0.1 mM to PLs, resulting in the partial liberation of PLs from Z-disks. We conclude, therefore, that the liberation of PLs by the binding of 0.1 mM calcium ions was the main cause for Z-disk weakening during the post-mortem aging of chicken.
We studied the effect of protease inhibitors at a high concentration on connectin and nebulin filaments in myofibrils. Calpastatin domain I at 0.1 mM bound to connectin and nebulin filaments, and deteriorated their physico-chemical properties; the calcium-binding ability of connectin and nebulin filaments was suppressed, the susceptibility of both filaments to trypsin was markedly decreased, and the resting tension of mechanically skinned fibers was increased by 2.5 times that of the control at a sarcomere length of 3.6 μm. This indicates that the connectin filaments were made more rigid. The same phenomenon was observed from the treatment of skinned fibers with 1 mM leupeptin whose resting tension was increased to 2 times the control value. Microscopically, both protease inhibitors induced dense aggregation and disappearance of the regular striation of myofibrils due to their non-specific binding to many myofibrillar proteins. The use of excess calpastatin domain I and leupeptin should therefore be avoided in physiological and biochemical studies on connectin and nebulin filaments, as well as on myofibrils.
The precipitate formed by ultracentrifuging a defatted soybean extract at 200,000×g for 50 min at pH 7.5 was composed of particles of 100-200 nm in diameter and enriched with 34-kDa, 24-kDa and 18-kDa proteins. An SDS-PAGE analysis showed these proteins to migrate to a position identical to that of oil-body-associated proteins (OBAPs; Herman, Planta, 172, 336-345, 1987).1) They were recovered in the precipitate of soy protein with 30-40% saturated ammonium sulfate in the presence of 10 mM 2-ME. The lipid composition of the precipitate by a TLC analysis showed that most of the polar lipids in the soybean extract had been condensed in the fraction, suggesting the association between OBAP and the polar lipids. Removal of OBAP and the polar lipids from the soybean extract by conventional centrifugation (10,000×g for 10 min) in the presence of 30 mM Na2SO4 and 30 mM CaCl2 at pH 2.8 was achieved with concomitant improvement of the volatile off-flavor. A soy protein isolate (SPI) prepared from such a soybean extract contained far fewer volatile off-flavor compounds than normal SPI did.
The small intestinal sucrase activity in a senescence-accelerated strain of mouse, SAMP1, was significantly lower than that in other strains, including its control strain, SAMR1. In contrast, the activity of isomaltase, which usually associates with sucrase to form a complex enzyme (SI complex), in SAMP1 was comparable to that in other strains. Thus, the ratio of the sucrase to isomaltase activities (S/I ratio) in SAMP1 was very low (about 0.15), compared with that in other strains (around 0.7). The S/I ratio in SAMP1 was abnormally low, even at a young age, indicating that senescence did not result in the low sucrase activity. Western blot analysis suggests that a large part of the isomaltase subunit occurred alone without the association of the sucrase subunit in this strain. In contrast, Northern blot analysis shows that the level of mRNA for the SI complex in SAMP1 was comparable to that in SAMR1. When the pancreatico-biliary ducts were ligated in SAMP1 to reduce the level of pancreatic proteases, a remarkable increase was observed in the sucrase activity, whereas the isomaltase activity was increased to a much smaller extent. This marked increase in sucrase activity resulted in the S/I ratio increasing to 0.84 18 h after the ligation. These results suggest the sucrase subunit of the SI complex to be abnormally unstable against pancreatic proteases in SAMP1.
We found the mechanism in flavonoids that can strongly suppress the mutagenicity of one of the food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The antimutagenicity was evaluated by IC50 value, the amount required for 50% inhibition of the mutagenicity of 0.1 nmol Trp-P-2, with Salmonella typhimurium TA98 strain in the presence of S9 mix. The flavones and flavonols were two orders stronger as antimutagens than such antimutagenic phytochemicals as chlorophylls and catechins. We had previously found flavonoids to be a desmutagen to neutralize Trp-P-2 before or during attack of DNA, because they had no effect on either the ultimate mutagenic form of Trp-P-2 (N-hydroxy-Trp-P-2) or the mutated cells. The desmutagenicity of the flavonoids did not depend on the hydroxy number or position that should be associated with antioxidative potency, and was also unaffected by the solubility of Trp-P-2 in the assay solution. The inhibitory effect of the flavonoids on the metabolic activation of Trp-P-2 to N-hydroxy-Trp-P-2 was almost in parallel with the antimutagenic IC50 value, when determined with a Saccharomyces cerevisiae AH22 cell simultaneously expressing both rat cytochrome P450 1A1 and yeast reductase. The Ki values of flavones and flavonols for the enzyme were less than 1 μM, while the Km value of Trp-P-2 was 25 μM. The antimutagenicity of the flavones and flavonols was thus concluded to be due to inhibition of the activation process of Trp-P-2 by P450 1A1 to the ultimate carcinogenic form. They were also able to act as antimutagens toward other indirect mutagens that were activated by P450 1A1.
The oxidation of trans-2-nonenal, the main compound causing staleness in beer, with a membrane fraction of acetic acid bacteria was investigated. For use of the fraction, it was necessary to inactivate membrane-bound alcohol dehydrogenase (ADH) first to avoid oxidation of alcohol, while maintaining membrane-bound aldehyde dehydrogenase (ALDH) activity. ADH was completely inactivated by treatment of the membrane fraction at 60°C for 15 min, without any loss of ALDH activity. ALDH activity was not affected by heat treatment even in the presence of 10% alcohol. Results of HPLC showed that a fraction incubated at 60°C for 15 min oxidized 50 ppb trans-2-nonenal added to a commercial beer sample without affecting the alcohol content. Our sensory panel found that the stale odor of trans-2-nonenal disappeared when the membrane fraction was so treated, with the resulting odor better than that of beer with trans-2-nonenal added but not treated with the fraction.
Magnetite prepared by an enzyme-dependent reaction gradually released iron ion into the acidic-to-neutral buffer solution. A preparatory experiment was performed to examine the efficiency of magnetite as an iron supplement. Feeding exsanguinated rats with being magnetite resulted in the hematocrit value being recovered without any serious adverse effect on the digestive organs.
A methanol extract from yellow mustard seeds had antibacterial activity against Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus. Two compounds with such activity were isolated from the extract. By instrumental analysis, the compounds were identified as 4-hydroxy-3-nitrophenylacetic and sinapic acids. Examination of the structure-activity relationship showed that the hydroxyl and nitro groups of the first compound were involved in the activity against all three species. The two methoxyl groups and the hydroxyl group in sinapic acid were effective against E. coli and all of the substituents of the benzene ring were effective against S. enteritidis. The presence of the propenoic group of the second compound was effective against S. aureus.
We developed a method to measure the amounts of antioxidative polyphenols and ubiquinones incorporated into the lipid bilayers of liposomes to estimate their affinities for cell membranes. Results were expressed in terms of an “affinity factor”, calculated by division of the amount of compound incorporated by the amount added to the liposomal solution. The results reflected dose-dependence of the biological activities of the compound found in earlier in vitro experiments with mammalian and bacterial cells.
A compound showing antimicrobial activity was isolated from an oil-macerated garlic extract by silica gel column chromatography and preparative TLC. On basis of the results of NMR and MS analyses, it was identified as Z-4,5,9-trithiadeca-1,6-diene-9-oxide (Z-10-devinylajoene; Z-10-DA). Z-10-DA exhibited a broad spectrum of antimicrobial activity against such microorganisms as gram-positive and gram-negative bacteria and yeasts. The antimicrobial activity of Z-10-DA was comparable to that of Z-ajoene, but was superior to that of E-ajoene. Z-10-DA and Z-ajoene are different in respect of substitution of the allyl group by the methyl group flanking a sulfinyl group. This result suggests that substitution by the methyl group would also be effective for the inhibition of microbial growth.
Cellulose production from sucrose by Acetobacter strains is accompanied by the accumulation of a water-soluble polysaccharide, called levan. To improve cellulose productivity, a levansucrase-deficient mutant, LD-2, was derived from Acetobacter strain 757 and used as a host for the construction of recombinant strains. An LD-2 mutant harboring a plasmid containing the sucrase gene, sucZE3, from Zymomonas mobilis together with zliS, a gene that encodes a secretion-activating factor under the control of the Escherichia coli lac promoter, had sucrase activity and produced much cellulose and little levan in a medium containing sucrose. In addition, a mutant levansucrase gene, mutant sacB, from Bacillus subtilis, which encodes a protein with little levan-forming activity, was generated by site-directed mutagenesis and introduced into the LD-2 mutant. This introduction also resulted in the higher cellulose productivity and little levan.
To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 188.8.131.52) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.
To produce a large amount of recombinant proteins in Escherichia coli, we constructed a unique cis-acting expression system using a plant virus protease. This new expression system could directly produce recombinant proteins, that had a biologically active form. A gene of nuclear inclusion-a (NIa), which had a specific amino acid sequence, was fused with a foreign protein gene at the same protein reading frame. One of the NIa-specific cleavage amino acid sequences, Gln-Ala, was also contained at the protein-protein junction. In the case of human interleukin-11 (hIL-11), a 23-kDa specific signal band was obtained from recombinant bacteria. N-terminal sequencing of the 23-kDa protein showed that NIa specifically cleaved the fusion protein at Gln-Ala, producing Ala-hIL-11. Furthermore, we could produce the mature rhIL-11 by extending the culture time. This 23-kDa protein had the same biological activity as hIL-11 in a mouse plasmacytoma, T1165. Combined with fermentation control, we produced mature rhIL-11 in E. coli.
Lactococcus lactis IO-1 was able to use xylose as a carbon source for nisin Z production; the yield based on sugar consumption was about 20% superior to that made with glucose under the same fermentation conditions. The optimal conditions for nisin Z production were with 4% xylose at pH 6.0 and 37°C. Addition of 0.1 M CaCl2 increased nisin Z production specifically, but not cell growth, acid production, or xylose consumption, and resulted in the maximum nisin Z activity of about 1.5 times that without CaCl2.
A microbe producing a protease with strong thermostability that was released extracellularly was isolated from soil. The isolate, MIB001, grew at from 15 to 51°C and pH 5.1-8.8 and was tentatively identified as a strain of Bacillus brevis. Rabbit antisera raised against a pure preparation of the protease did not cross-react with thermolysin or neutral metalloprotease from Bacillus stearothermophilus KP1236.
A structural gene of Tri101, which encodes trichothecene 3-O-acetyltransferase, was isolated as a 3-kb XhoI-XbaI fragment from the trichothecene producer Fusarium graminearum strain F15. The gene contained no introns, and the coding region was 0.7-kb downstream of a putative UTP-ammonia ligase gene which obviously is not related to the biosynthesis of trichothecenes. Tri101 was expressed when T-2 toxin was added, but this induction was not dependent on the expression level of Tri6, a transcription activator gene in the trichothecene biosynthetic and regulatory gene cluster.
Fourty-two kinds of benzaldehyde O-alkyloximes derived from benzaldehydes were prepared and their biological activities were investigated. Introduction of a fluorine or bromine atom to the benzene ring of the oximes enhanced their phytotoxic activity. The O-alkyloximes with a fluorine atom at the 3 or 4 position of the benzene ring were more active than the other oximes in the GA3-induced α-amylase induction inhibition test. In the transpiration test, 4-bromobenzaldehyde O-carboxylmethyloxime was the most active. The O-alkyloximes exhibited weak abscisic acid-like activity by inhibiting not only the germination, root growth and transpiration of higher plants but also GA3-induced α-amylase induction in embryoless barley seeds.
Aminorhodanine (1) showed strong insecticidal activity against Culex pipiens pallens and Musca domestica, with respective LD50 values of 0.21 μg/insect and 0.87 μg/insect. Compound 1 had antifungal activity against Aspergillus niger ATCC-16404, Trichophyton mentagrophytes IFO-32412, Candida albicans ATCC-10231, Hansenula anomala OPS-308 and Penicillium expansum IFO-8800. In particular, 1 had potent antifungal activity against Aspergillus niger ATCC-16404, its minimal inhibitory concentration (MIC) being 6.25 μg/ml. Both activities of 1 were much higher than those of rhodanine (4), suggesting that the introduction of an amino group into N-3 of 4 plays an important role in the biological activity of rhodanine-related compounds. On the other hand, N-acetylaminorhodanine (2) and N-benzoylaminorhodanine (3) did not show either activity, suggesting that the free amino group at N-3 of 1 is closely related to the inhibitory activity of rhodanine derivatives.