We determined the anti-cataract effects and antioxidative activities of four 4-hydroxy-3(2H)-furanones. These four furanones showed similar antioxidative activities in the ferric ion reduction model. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone (EHMF) exhibited a higher suppression effect on lipid peroxidation in human plasma than the other furanones did. The effects of hydroxy furanones on the onset of cataract in spontaneous cataract rat (ICR/f rat) were tested, and it was observed that HDMF and EHMF inhibited cataract formation. These results suggest that the antioxidative activity of HDMF and EHMF against superoxide radicals in lens tissue contributed to inhibiting the onset of spontaneous cataract.
Four new metabolites related to IAA (indole-3-acetic acid) were isolated from an aqueous methanol extract of rice bran and were elucidated by spectroscopic methods to be a pair of diastereomers of 5-O-β-D-glucopyranosyl 3,5-dihydroxy-2-indolinone-3-acetic acid (A-1 and A-2) and of 5-O-β-D-cellobiosyl 3,5-dihydroxy-2-indolinone-3-acetic acid (B-1 and B-2). Compound B-1 was also a diastereomer of compound B-2 at the C3 position with an asymmetric carbon atom on an indolinone skeleton, like the relationship between compounds A-1 and A-2.
Diapause hormone is a 24-amino acid peptide amide, and its C-terminal penta-peptide amide structure of FGPRL-NH2 is believed to be essential for biological activity. The penta-peptide amide, the shorter peptide amides, and their derivatives and analogs were prepared to determine the minimal structure for biological activity. The C-terminal amide group of the penta-peptide amide was not replaced with the other functional groups, but Gly, the 4th amino acid from the C terminal, could be substituted with an other amino acid while maintaining the biological activity. The shorter peptide amide, PRL-NH2, possessed low but significant activity, indicating the minimum structure of diapause hormone. By modifying its N-terminal, the aromatic ring of Phe is shown to enhance the activity of PRL-NH2.
Saponins in bulbs of a mutant of elephant garlic were investigated, and three new steroidal saponins named yayoisaponins A-C were obtained together with the known dioscin and aginoside. Their structures, including the relative stereochemistry, were elucidated by spectral data interpretation, while the absolute stereochemistry of the sugar moieties was assigned on the basis of a chiral gas chromatographic analysis of the acid hydrolysates. Yayoisaponins A-C and aginoside exhibited not only in vitro cytotoxicity against P388 cells at 2.1 μg/ml, but also antifungal activity against Mortierella ramanniana at 10 μg/disk.
Two minor components of solanapyrones D and E, phytotoxins from Alternaria solani, were isolated. The absolute stereochemistry of D and the optical purity of solanapyrones A and D were determined by chemical transformation and measurement of the CD spectrum. In addition, complete signal assignment of the 1H- and 13C-NMR spectra of solanapyrones A and D, and their solution conformations were studied. Feeding experiments with various isotopically labeled acetates and methionine established the biosynthetic pathway of solanapyrones. The phytotoxicity of solanapyrones on lettuce seedlings is discussed.
The new aldobiuronic acid, 3-O-α-D-glucopyranuronosyl-L-rhamnopyranose, was isolated from the acid hydrolyzate of an acidic polysaccharide in cells of Chlorella vulgaris K-22. Its structure was elucidated by NMR spectroscopic analyses.
A number of atropine analogs were synthesized and their effects on larval growth of the silkworm, Bombyx mori, were investigated by both topical application and dietary administration. Among the tested compounds, 8-methyl-8-azabicyclo[3.2.1]octan-3α-ol 2,2-diphenylpropionate (5), an antagonist of the muscarinic acetylcholine receptor in mammals, significantly prolonged the duration of the instar. When fed on compound 5 at 30 ppm, some of the larvae failed to molt. A 2,2-diphenylpropionate moiety was indispensable for this activity. Compound 5 had more potent activity than atropine which is known to inhibit PTTH release in vitro.
Nocardamine, a kind of siderophore, was isolated as an inducer of morphological changes in the insect cells, BM-N4. It changed the morphology of the cells from round to an unusual spindle shape. This activity of nocardamine was inhibited by adding the ferric ion.
Sphingomyelins and ceramides bearing a docosahexaenoyl or arachidonoyl group were synthesized from sphingosylphosphorylcholine and sphingosine, respectively, by dicyclohexylcarbodiimide-mediated acylation.
Amaranth belongs to a nutritious class of pseudocereals. The well balanced amino acid composition of amaranth compared with those of major cereals would indicate that it deserves a quantitative study of its chemical properties. This work was undertaken to compare Amaranthus (A.) caudatus with a number of other plants on the basis of the sequences of various proteins and the composition of their alcohol-soluble protein mixture and glutelins. Alcohol-soluble proteins were extracted with 55% isopropanol (2-ProOH)+5% 2-mercaptoethanol (2-ME) and glutelin fractions were obtained with borate buffer+3% 2-ME+0.5% sodium dodecyl sulfate (SDS), pH 10. Protein fractions were then electrophoreded on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). FASTA and TFASTA programmes were used for comparison of amino acid sequences. Dot matrix analysis and secondary structure predictions which were drawn by Plotstructure, were taken from the GCG package. Electrophoretic tests failed to indicate significant correlation between prolamins from cereals and other plants with the alcohol-soluble fractions from amaranth, proving that these proteins cannot represent the main fraction in amaranth. On the other hand, glutelins shared some common electrophoretic bands with other cereals and showed some identity by SDS-PAGE. Amino acid sequences of A. caudatus (100% identity) had degrees of similarity in the range of 71.4 to 52.2% with rice, garden pea, jobs’ tears, maize, and yam. Rice glutelin had similarity in the range of 93.3% to 44.8% with oats, soybean, and pea. Secondary structures of A. caudatus (using conservative amino acid replacement), jobs’ tears and rice glutelins, oat globulin, and pea legumin sequences were predicted. Some relationship was shown among electrophoretic patterns of alcohol-soluble proteins and glutelins of A. caudatus.
The budding yeast Saccharomyces cerevisiae was shown to have two kinetically distinguishable glutathione transport systems. While one with high affinity (GSH-P1; KT=0.045 mM) was regulated, the other with low affinity (GSH-P2; KT>2 mM) was not. GSH-P1 was highly specific to glutathione, and its activity was quickly lost by suspending the cells in buffer solutions. This activity loss was not observed if glucose-containing buffer was used. In addition, ρ- isolates had only about one half of the glutathione transport activity of the original (ρ+) strain. Therefore, it is concluded that GSH-P1 is an ATP-driven transport system. Strong and moderate inhibition of GSH-P1 by protonophores and ionophores, respectively, are attributed to competition for ATP between GSH-P1 and proton- and cation-pumps, respectively.
From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTPγS and [3H]-GDP with association constants of 1.5×106 M-1 and 0.58×106 M-1, respectively. The binding of [35S]-GTPγS was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTPγS and [3H]-GDP were exchanged with GTPγS most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
The 200-kb linear plasmid pSLA2-L was suggested to be involved in the production of lankamycin and lankacidin in Streptomyces rochei 7434AN4. In this study, we have constructed a physical map for 23 PstI fragments of pSLA2-L, the sum of which was 206 kb. Detailed restriction maps for both ends of pSLA2-L revealed the presence of terminal inverted repeats, the size of which was found to be 2.1 kb by cloning and sequencing of the end-points. Hybridization experiments using two polyketide biosynthetic genes, eryAI and actI, located their homologous regions on PstI fragments A and I, respectively.
A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (ε280 nm1%) of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50°C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.
The molecular structure of starch granules formed in suspension-cultured cells of Ipomoea cordatotriloba Denn. was characterized by its chain length distribution, which was compared to those of the starches from the root and leaf of the original plant. The cultured cell starches were spherical and had a very small granule size (about 2 μm). The debranched starches roughly separated into three fractions during gel-permeation chromatography, and the fractions were defined as Fr.1, 2, and 3, respectively. The chain length distribution of the debranched cultured cell starch showed that the high molecular weight fraction (Fr.1), referred to as an amylose fraction, was much less than those of the root and leaf starches. The ratio of the two lower fractions (Fr.3/Fr.2) of the cultured cell starch, which was mainly derived from unit chains of amylopectin, was greatest among the starches, suggesting that the amylopectin from the cultured cell starch has much shorter unit chains. By X-ray diffraction analysis, it was found that both cultured cell and leaf starch granules have low crystallinity.
ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.
The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).
The nirQOP operon, which is located between the genes for nitrite reductase and nitric oxide reductase in Pseudomonas aeruginosa, encodes a putative ATP-binding protein and two putative transmembrane proteins. Phylogenetic analysis showed that NirO belongs to the family of subunit III of cytochrome oxidases but is distantly related to the other bacterial or mitochondrial proteins. P. aeruginosa strains that lacked the nirOP genes had all enzyme activities for denitrification and could grow under anaerobic conditions with nitrate or nitrite as an electron acceptor. However, the energy conservation efficiency of anaerobic respiration was lower in these strains than in strains harboring the nirOP gene.
An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The Km and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.
Two Dictyostelium discoideum ribosomal protein genes, denoted DdL27a and DdL37a, were isolated and sequenced. The DdL27a gene contained an open reading frame of 148 amino acids coding for a putative 16,407 Da protein, which was similar to rat L27a (82.6% similarity) and to ribosomal proteins from other species. The gene contained a 311-bp intron downstream from the ATG initiation codon with an A+T content of 75%. The DdL37a gene encoded a 9,999 Da protein consisting of 91 amino acids, which had high sequence similarity to rat, human, and chicken ribosomal protein L37a, and was interrupted by two introns of 254 bp and 75 bp in length. The DdL37a protein contained a typical zinc finger motif (Cys-X2-Cys-X14-Cys-X2-Cys), which may be involved in the interaction of proteins with nucleic acids. Genomic DNA blot analysis indicated that the DdL27a and DdL37a genes are present in single copies in the Dictyostelium haploid genome. The DdL27a and DdL37a mRNA were expressed maximally in growing amoebae, and their levels decreased during multicellular development, coordinately with the observed decrease in ribosome accumulation during later development.
Barbatic acid, a lichen-derived depside, inhibited oxygen evolution in spinach thylakoid membranes. It also affected parameters of Chl fluorescence, (Fm′-F)/F and Fv/Fm. Using specific donors and acceptors of electrons, we found two sites of inhibition in the PS II complex. The primary site, which is responsible for the inhibition of oxygen evolution, is at the reducing side of QA, possibly at QB. The other site is at the oxidizing side of P680 but not in the oxygen evolving complex, suggesting Yz as the target. At both sites, irreversible binding of the depside to the targets seems to be responsible for the inhibitions. Among the 8 lichen acids compared, barbatic acid was the most potent inhibitor for both the reducing site and oxidizing site.
A feruloylesterase was purified from the extracellular broth of Aspergillus awamori grown on wheat bran culture. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent Mr of 35,000 and a pI of 3.8, respectively. The substrate specificity of the purified enzyme differed obviously from that of acetylesterase of A. awamori. The enzyme bound to microcrystalline cellulose.
Aqualysin I is an alkaline serine protease isolated from Thermus aquaticus YT-1, an extreme thermophile. We have measured the P1-specificity of aqualysin I, using wild-type and five P1-substituted derivatives of Streptomyces subtilisin inhibitor (SSI). SSIs efficiently inhibited the activity of aqualysin I, with low substrate specificity. Charge and hydrophobicity of side chain of the P1 amino acid residue showed no significant effect to the P1-specificity of this enzyme.
Human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) was phosphorylated by incubation with [γ-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Approximately 0.8 mol of phosphate per mol subunit of HP2K was incorporated by either PKA or PKC. However, with additional incubation with PKA following incubation with PKC or vice versa, no additional phosphate was incorporated into the HP2K. The phosphorylation sites for the two protein kinases were identified by peptide mapping and microsequencing following digestion of phosphorylated-HP2K with clostripain. Evidence is also suggested for a common phosphorylation site (Ser-460) for PKA and PKC.
Single-chain monellin, a sweet protein, was synthesized by stepwise solid-phase synthesis. After deprotection, and purification with HPLC, single-chain monellin was obtained in a yield of 0.3% based on the starting resin.
A brown-colored fraction containing heme was separated from the lipoprotein-free plasma of Japanese eel (Anguilla japonica). This fraction prevented the oxidation of very-low-density lipoprotein incubated with CuSO4 ranging in concentration from 0.5 to 3 μM. The antioxidant activity seemed to derive from the heme-binding proteins of the brown-colored fraction.
The enzymatic properties of phytolacain G, a protease isolated from green fruit of pokeweed, were compared with those of phytolacain R, a protease obtained from ripe fruit. The optimum pH of phytolacain G was 7.5-8.0 at 37°C using casein as the substrate. The enzyme was strongly inhibited by iodoacetic acid and p-chloromercuribenzoic acid, but not by diisopropyl fluorophosphate or EDTA. These results indicated that phytolacain G was a cysteine protease, like phytolacain R. Nine sites of oxidized insulin B-chain were cleaved by phytolacain G during 20 h of hydrolysis. The six sites cleaved by phytolacain G were also cleaved by phytolacain R. The substrate specificity of phytolacain G was broad, but the preference for hydrophobic residues at the P2 position was similar to the substrate specificity of papain. The amino-terminal sequence of phytolacain G was not identical with that of phytolacain R; however, the amino acid residues conserved in the papain family were also conserved in this enzyme.
The anti-lipolysis by ginseng polypeptide and its modified peptides was examined using porcine adipose cells. Ginseng polypeptides modified by amino acid substitution or proteolyzation reduced or lost the inhibiting activity of adrenalin-induced lipolysis. Correlation between the anti-lipolytic activity of ginseng polypeptide and its Mg2+- and ribose-binding activities is discussed.
Topoisomerase II from human placenta was strongly inhibited by prostaglandins such as Δ12,14-PGJ2, Δ12-PGJ2, PGA2, and PGA1, which have an antitumor activity. The topoisomerase II inhibitions by these prostaglandins were dose-dependent and IC50 values of Δ12,14-PGJ2, Δ12-PGJ2, PGA2, and PGA1 were 22 μM, 74 μM, 75 μM, and 98 μM, respectively. Topoisomerase I from calf thymus gland was inhibited by Δ12,14-PGJ2, Δ12-PGJ2, and PGA2, but not inhibited by other prostaglandins even at high concentrations. Only the prostaglandins having the antitumor activity inhibited topoisomerase II. The results suggest that the antitumor activity of prostaglandins may be related, at least in part, to inhibition of topoisomerase II in tumor cells.
An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.
An aqueous suspension of bacterial cellulose (BC) has such physical properties as higher viscosity, emulsion-stabilizing effect and filler retention than cellulose of other origins. The specific surface areas of BC, microfibrillated cellulose and wood pulp were evaluated by determining the maximum amounts of adsorption of Congo red, cellobiose dehydrogenase (CDH) and xyloglucan. There was a positive linear correlation between the above-mentioned physical properties of each cellulose sample and the specific surface area derived from the maximum amount of CDH adsorbed. The highest physical property values for BC result from the largest external surface area of the fibrils of BC to which CDH was adsorbed.
The hypoglycemic effect of water-soluble polysaccharide(FA) from fruiting bodies of Auricularia auricula-judae Quel. was investigated on genetically diabetic mice (KK-Ay) from 10 to 14 weeks of age. Male mice were divided into 3 groups,the control group and FA-fed group having free access to the control diet or FA diet (30 g of FA/kg of diet). The food-restricted group had restricted access to the control diet at the level of the diet consumed by the FA-fed group. Compared with the control group, FA supplementation had a significant effect in lowering plasma glucose, insulin, urinary glucose, and food intake. FA administration also increased the tolerance to intraperitoneal glucose loading and the hepatic glycogen content. In the food-restricted group, the reduced food intake slightly lowered the plasma and urinary glucose levels, but did not improve hyperinsulinemia and glucose tolerance. This study shows that FA had a hypoglycemic effect on KK-Ay mice, and the reduced food consumption was not a major factor which contributed to the hypoglycemic action of FA.
The antioxidative activity of α-tocopherol and Trolox, a water-soluble carboxylic acid derivative of α-tocopherol, in an emulsion stabilized with the hydrophilic emulsifier, polyoxyethylene (20) sorbitan monolaurate, was evaluated by measuring the changes in linoleic acid content, peroxide value, and thiobarbituric acid value. The activity of α-tocopherol and Trolox to depress the autoxidation of linoleic acid was stronger when they were added in the aqueous phase than when added in the oil phase, and Trolox in the aqueous phase was the most effective. The results of interfacial-tension measurements support the idea that the affinity of phenolic antioxidants to the surface layer of oil droplets might be related to their antioxidative activity. The antioxidative activity of α-tocopherol and Trolox added in the oil phase was markedly enhanced by using β-lactoglobulin together with Tween 20 to stabilize a linoleic acid emulsion. The results of a linoleic acid determination with cold and radioactive linoleic acid indicate that linoleic acid in a complex with the β-lactoglobulin molecule would be effectively protected by the phenolic antioxidants, α-tocopherol and Trolox, against autoxidation.
We examined the effect of three dietary fats, safflower oil (SAF) rich in linoleic acid, borage oil (BOR) rich in γ-linolenic acid, and perilla oil (PER) rich in α-linolenic acid, on the lipid metabolism, and chemical mediator and immunoglobulin levels in Sprague-Dawley rats, as well as the dietary effect of sesame-derived antioxidative sesamin. The serum cholesterol, phospholipid, triglyceride, prostaglandin E2 level and splenic leukotriene B4 level were lower in the rats fed on BOR or PER than in those fed on SAF. SES feeding suppressed the expression of the lipid-decreasing effect of BOR, but not in the rats fed on PER. In respect of the fatty acid composition of the liver and spleen, PER feeding gave a lower arachidonic acid level, and higher eicosapentaenoic and docosahexaenoic acid levels than SAF feeding did, while the effect of BOR feeding was marginal. The effect of SES feeding on fatty acid composition was much smaller than that of dietary fats. In respect of immunoglobulin production, PER+SES feeding gave the lowest IgE productivity in the mesenteric lymph node lymphocytes. These results suggest that PER feeding regulated lipid metabolism and exerted an anti-allergic effect by a different mechanism from that with BOR feeding.
The immediate response of protein degradation to food intake and the factors for its regulation in rat skeletal muscle were examined. The concentration of Nτ-methylhistidine (MeHis) in serum and the rates of MeHis release from isolated soleus and extensor digitorum longus muscles were reduced in the period from 3 to 6h after refeeding, indicating that the rate of myofibrillar protein degradation in the rat decreased immediately after refeeding. Changes in the serum concentration of insulin and corticosterone were not synchronized with those in the myofibrillar protein degradation. When rats were fed on a protein-free diet, no reduction of serum MeHis concentration or of the rate of MeHis release from isolated muscles after refeeding was apparent. Furthermore, there was a tendency toward suppressing myofibrillar protein degradation with a higher protein content of the diet. These results suggest that the suppression of myofibrillar protein degradation by food intake was regulated by dietary proteins.
The relationships between 4-hydroxy-2-nonenal (HNE), 2-thiobarbituric acid reactive substances (TBARS) and n-6 polyunsaturated fatty acids (n-6 PUFAs) were investigated for pork stored at 0, -20 and -80°C. A significant linear correlation was apparent between HNE and TBARS for the pork stored at each temperature. However, the n-6 PUFA content remained unchanged during storage.
The cyanoglycoside, prunasin, was isolated for the first time as an aroma precursor of benzaldehyde from fresh tea leaves (Camellia sinensis var. sinensis cv. Yabukita). Prunasin was readily hydrolyzed by a crude enzyme prepared from the fresh tea leaves to liberate benzaldehyde. The isomerization of prunasin under neutral conditions was also recognized.
The expression of mRNA of leptin, the product of the obese gene, in bovine adipose tissue was analyzed by a lysate RNase protection assay. The mRNA level was significantly decreased by food deprivation for two days and partially recovered after 3 hr of refeeding, indicating that obese gene expression in the ruminant was regulated by feeding.
N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.
The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery lager yeast have one ATF2 gene, while S. bayanus has no similar gene.
Sixteen marine methanotrophic bacteria were isolated and 14 marine methanotrophic mixed cultures were obtained. They were assayed for soluble methane monooxygenase (sMMO) by naphthalene oxidation and only one isolate (strain NI) was positive. Strain NI degraded trichloroehylene (TCE) more efficiently than other methanotrophic isolates containing no sMMO only under copper limiting conditions. Dimethyl sulfide (DMS), one of the radiatively important trace gases released from the sea, was transformed to dimethyl sulfoxide (DMSO) by methanotrophs and the efficiency for the transformation of DMS to DMSO was not as much affected by the presence of sMMO as that of TCE. The taxonomical properties of strain NI and phylogenetic analysis based on 16S rDNA genes indicated that strain NI was a type I methanotroph belonging to the genus Methylomicrobium, and closely related to Methylomicrobium pelagicum. The partial mmoX gene of strain NI was amplified by the primers common to three other mmoX genes and its 270 bp were sequenced. 77 residues out of the 89 amino acids derived from the sequences were common among the four mmoX genes.
We isolated two extracellular β-glucosidases (EX-1: 145 kDa, EX-2: 130 kDa) and one cell wall bound β-glucosidase (CB-1: 120 kDa) from Aspergillus kawachii and characterized their physical and kinetic properties. From the results of N-terminal amino acid sequence, enzymatic parameters and deglycosylation of the three purified enzymes, we strongly suggest that these three enzymes were products of the same gene, modified by different degree of glycosylation. All three purified β-glucosidases adsorbed to the purified cell wall fraction of this strain. This association could dramatically improve the stability of purified enzymes. These three purified β-glucosidases were readily inactivated even in moderate conditions but became very stable upon the addition of the purified cell wall fraction. The all purified β-glucosidases were stable in the range of pH 2.0-9.0 and stable below 30°C with 2 mg/ml of the purified cell wall fraction.
Some methylotrophic yeasts produce methyl formate from methanol and formaldehyde via hemiacetal formation. We investigated Saccharomyces cerevisiae to find whether this yeast has a carboxylate ester producing pathway that proceeds via hemiacetal dehydrogenation. We confirmed that the purified alcohol dehydrogenase (Adh) protein from S. cerevisiae can catalyze the production of esters. High specific activities were observed toward the hemiacetals corresponding to the primary alcohols when ether groups were substituted for methylene groups, resulting in the formation of formate esters. Both ADH and methyl formate synthesizing activities were sharply reduced in the Δadh1Δadh2 mutant. The ADH1 and ADH2 genes encode the major Adh proteins in S. cerevisiae. Thus, it was concluded that the S. cerevisiae Adh protein catalyzes activities for the production of certain carboxylate esters.
The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a598, b562(558), and c550 as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa3 terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H+/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.
Tetragenococcus halophila is a Gram-positive halophilic lactic acid bacterium used for soy sauce fermentation. We isolated a mutant, T. halophila 3E4, triply defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase. 3E4 selectively metabolized pentoses such as xylose and arabinose in the presence of hexoses such as glucose and galactose. We present here an example of the metabolic engineering of catabolite control.