An efficient solid-phase synthesis of Fmoc (glyco)peptide thioesters is described. Fmoc·Ser·OAll and Fmoc·Thr·OAll bound to resin with a silyl ether linker were deallylated by Pd(0) catalysis and condensed with thiophenol, benzyl mercaptane, and ethyl 3-mercaptopropionate by activation with DCC/HOBt. The thioesters were released from the resin either by treatment with CsF-AcOH or by acidic hydrolysis. The effectiveness of this silyl linker strategy is further demonstrated by the synthesis of more complex (glyco)peptide thioesters 25, 26 and 27 involving N→C and C→N peptide elongation.
The new caffeic acid derivative, subulatin (1), was isolated from in vitro cultured liverworts, Jungermannia subulata, Lophocolea heterophylla, and Scapania parvitexta. The structure of 1 involved two caffeic acids, D-glucose, and 2-carboxy-6-(1,2-dihydroxy-ethyl)-4,5-dihydroxy-5,6-dihydro-4H-pyran. The connectivity of those and the absolute stereochemistry of 1 were elucidated on the basis of spectroscopic evidence. The antioxidative activity of 1 was comparable to that of α-tocopherol. (2′R)-Phaselic acid (2a) and (−)-9,2″-epiphylloyl-L-malic acid (4) were also isolated from J. subulata and L. heterophylla, respectively. A chiral HPLC analysis of the p-bromobenzoyl-malic acids derived from 2a showed that 2a from J. subulata was unusual (+)-trans-caffeoyl-D-malic acid.
The effect of the surfactant, α-[4-(1,1,3,3-tetramethylbutyl) phenyl]-ω-hydroxypolyoxy-1,2-ethanediyl, on the adsolubilization of cholesterol and/or dotriacontane as model compounds of the epicuticular wax of mature tomato (Lycopersicon esculentum Mill.) fruit was investigated. Cholesterol as a model compound of such triterpenols as α- and β-amyrins was solubilized in a concentration-dependent manner above the critical micelle concentration (cmc), while non-detectable quantities of the saturated hydrocarbon, dotriacontane, was solubilized at any concentration used. However, the surfactants solubilized more cholesterol from mixed than single membranes. The surfactants with a shorter polyoxyethylene (POE) chain length solubilized greater quantities than those with longer POE chains, suggesting that the microenvironment of micelles related to the polyoxyethylene moiety had an important effect on surfactant solubilization and that the octylphenol moiety must be capable of adsorbing to a specific region of the reconstituted membrane like dotriacontane.
Optically active α-methylbenzyl phenyl ureas (MBPUs) show diverse plant physiological properties. Experiments were conducted to evaluate the salt-stress response of just-germinated rice seedlings supplemented with the S-enantiomer of MBPUs by assessing the growth and Na+ content. This study indicates that S-MBPUs served as a unique stress reliever for just- germinated young seedlings of rice injured by salinity. NaCl severely affected the root growth of rice seedlings. Concomitant treatment with S-MBPUs effectively ameliorated the growth inhibition of rice by NaCl. Glycine betaine (GB) did not act as a reliever of the NaCl stress. The addition of S-α-methylbenzyl 2-fluoro-4-methylphenyl urea (7, denoted as S-FM) to the saline medium ameliorated not only the root growth but also the protein content and dry weight of roots depending upon its concentration. The protein content, Na+ content and growth rate were correlated to each other with a positive relationship. The Na+ distribution ratio (S/R(Na+)) between the shoot and root increased with increasing concentration of NaCl when added alone, viz. with increasing growth reduction. A concomitant treatment with S-FM (7), however, resulted in the S/R(Na+) value becoming smaller with growth amelioration. This indicates that S-FM (7) controlled the translation of Na+ from the roots to shoots. S-FM (7) would have influenced some inherent functions connected with the Na+ behavior in the rice plant, although details of the mechanism for normalization of the S/R(Na+) ratio are still not clear.
A series of N-substituted aryl and alkyl carbamates (RNHCOOR′; R: aryl, alkyl; R′: aryl, alkyl) was prepared and screened for inhibitory activity toward the germination of oat seeds. The activity of each compound was compared with that of chlorpropham (isopropyl 3-chlorocarbanilate). Some of the synthetic carbamates possessing the N-(phenylthio)methyl group, PhSCH2NHCOOR´, showed inhibitory activity close or comparable to that of chlorpropham.
The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35–37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E. coli, Gluconobacter oxydans, and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.
The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50°C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.
We cloned a complete cDNA encoding rye seed chitinase-c, designated RSC-c, by rapid amplification of cDNA end and PCR procedures. The cDNA of RSC-c consists of 1,018 nucleotides and includes an open reading frame encoding a polypeptide of 266 amino acid residues. A recombinant RSC-c was produced by expression in Escherichia coli Origami(DE3) and purified. rRSC-c had almost the same chitinase activity toward glycolchitin and antifungal activity against Trichoderma sp. as the authentic RSC-c did. RSC-c mutants were subsequently constructed and characterized with respect to their chitinase and antifungal activities. Mutation of Glu67 to Gln completely abolished the chitinase activity and diminished the antifungal activity. Considerable decreases in both activities were observed in the mutations of Trp72 and Ser120 to Ala, and Glu89 to Gln. The roles of these residues in the catalytic event of RSC-c are discussed.
Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and γ-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K136I137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [γ-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N- [2(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K136I137S138 of LukS-I is important for the leukocytolysis of HPMNLs.
We earlier isolated a cDNA clone (NGR1) encoding a wound-inducible ribonuclease (RNase NW) from leaves of Nicotiana glutinosa [Kariu et al. Biosci. Biotechnol. Biochem., 62, 1144–1151 (1998)]. In this study, two distinct cDNA clones, NGR2 and NGR3, encoding proteins with a ribonuclease-related sequence in the N. glutinosa leaves, were amplified and sequenced. The nucleotide sequences of NGR2 and NGR3 consist of 1244 bp and 1069 bp, and have open reading frames encoding 277 (RNase NGR2) and 236 (RNase NGR3) amino acid residues, respectively. The deduced amino acid sequences of the putative RNases NGR2 and NGR3 showed 33% and 58% amino acid sequence identity, respectively, with that of RNase NW and 32% identity with each other. Sequence comparison showed that NGR2 is similar to RNase RNS2 (61%) from Arabidopsis thaliana, while NGR3 is related to RNase LX (84%) from tomato (Lycopersicon esculentum). RNA gel blot analysis showed that the RNase NGR2 gene is constitutively expressed to measurable levels; it is not increased by either wounding or TMV infection. In contrast, the expression of the NGR3 gene is induced after 48 h upon TMV infection.
A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a β-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.
An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was purified and characterized. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 60°C, respectively. The gene (mprI) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed significant similarity to metalloproteases classified into the thermolysin family. Furthermore, sequence analysis showed that another metalloprotease (MprII)-encoding gene was located downstream from mprI. The deduced amino acid sequence of MprII showed high similarity to metalloproteases of the aminopeptidase family. Similar repeated C-terminal extensions were found in both MprI and MprII.
The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70°C, and the reaction was optimal around 70°C, as well as native ABN-TS.
The gene encoding α-L-arabinofuranosidase (STX-IV), located upstream of the previously reported stxI gene, was cloned and sequenced. The gene is divergently transcribed from the stxI gene, and the two genes are separated by 661 nucleotides. The stxIV gene consists of a 1,092-bp open reading frame encoding 363 amino acids. The deduced amino acid sequence of the gene showed that STX-IV was an enzyme consisting of only a catalytic domain, and that the enzyme had significant similarity with α-L-arabinofuranosidases belonging to family 62 of glycosyl hydrolases. The stxIV gene was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Arabinoxylan and oat spelt xylan were good substrates for STX-IV, however, the enzyme showed a low activity with p-nitrophenyl α-L-arabinofuranoside. The optimum pH and temperature were 5.0 and 60°C, respectively.
The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovora subsp. carotovora Er are induced by mitomycin C. Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent. The optimal temperature for Pnl production was 30°C. However, the optimal temperature for both Ctv production and cell lysis was 23°C, at which Pnl production was reduced to 47% of the maximum. These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgB genes.
A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.
Heterocapsa circularisquama (Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquama showed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama. Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquama but also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.
All cysteines of mouse ileal and hepatic sodium-dependent bile acid transporters (Isbt and Ntcp, respectively) were individually replaced by alanine. Replacement of Cys106 in Isbt and Cys96 in Ntcp, which are located closely in alignment, decreased taurocholate uptake. Although Cys51 in Isbt is conserved in Ntcp, the replacement spoiled Isbt only. Both similarity and difference in the arrangement of functional sites are suggested.
We have reported that the chitinolytic system of Alteromonas sp. strain O-7 consists of chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), β-N-acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB, and GlcNAcaseC), and a novel transglycosylative enzyme (Hex99). The gene encoding a β-hexosaminidase with an unusual substrate specificity (hex86), located upstream of the hex99 gene, was cloned and sequenced. The gene encoded a protein of 761 amino acids with a calculated molecular mass of 86,758 Da. The deduced amino acid sequence of Hex86 showed sequence similarity with β-hexosaminidases belonging to family 20. The hex86 gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme rapidly cleaved p-nitrophenyl- β-N-acetyl-D-glucosaminide and slowly cleaved p-nitrophenyl-β-N-acetyl-D-galactosaminide. Unexpectedly, the enzyme did not hydrolyzed chitin oligosaccharides under the assay conditions for synthetic glycosides. However, after prolonged incubation with excessive quantities of the enzyme, Hex86 hydrolyzed chitin oligosaccharides. These results indicate that Hex86 is a novel enzyme that prefers p-nitrophenyl-β-N-acetyl-D-glucosaminide to chitin oligosaccharides as a substrate.
An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.
Cell morphology and cell-to-cell adhesion of taste bud cells were significantly altered by extracellular Ca2+ during in vitro culture. Under high Ca2+ condition (above 0.5 mM), the cells were tightly associated with each other and formed packed aggregates. Under low Ca2+ condition (below 0.1 mM), the cells were dispersed and had an elongated shape. These two forms were reversible and specifically dependent on Ca2+. The results indicate that extracellular Ca2+ regulates cell shape and cell-to-cell adhesion of taste bud cells.
We have previously reported that water-soluble β- glucan completely devoid of mannoprotein and purified from the yeast cell wall effectively stimulated the macrophage function (Biosci. Biotechnol., Biochem., 65, 4, 837-841 (2001)). In this present study, to increase the yield of water-soluble β-glucan, the wild type of Sacharomyces cerevisiae, JH, was treated with a combination of UV irradiation and laminarinase (endo-β-(1,3)-glucanase) to yield the laminarinase-resistant mutants, JUL1 and JUL3. Water-soluble β-glucans that were free of mannoprotein from JH, JUL1 and JUL3 were purified and their effects on TNF-α secretion and phagocytosis by macrophages were evaluated. Crude β-glucan was first solubilized from the yeast cell wall by alkaline extraction and then subjected to an acid treatment. The residual mannoprotein was completely removed by DEAE and ConA chromatography. The yield of water-soluble β-glucan in both mutants (JUL1, 5.11%; JUL3, 5.76%) was about 5-fold higher than that of the wild type (1.16%). The water-soluble β-glucan from JH induced TNF-α secretion slightly more than that from JUL1 or JUL3: TNF-α secretion by JH at 50, 200, 500 μg/ml of β-glucan was 11-17% more than that by JUL1 or JUL3 for the same treatment. β-Glucan from the wild type stimulated phagocytosis slightly more than that from the mutants. These mutants could therefore effectively produce purified water-soluble β-glucan with immune activity.
Spinach leaves were found to contain two potent antitumor promoters as detected by the activity against tumor promoter-induced Epstein-Barr virus activation. The active components were identified as 1-O-α-linolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3- O-β-D-galactopyranosyl-sn-glycerol and 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol by spectroscopic data and some chemical and enzymatic reactions. Their contents significantly varied with the cultivar and with the culture conditions.
Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 μl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 Å molecular sieves at 25°C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6–C18) were synthesized at 64–86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.
The purpose of this study was to determine whether the quantity and quality of dietary protein affected the polysome profile of the brain in aged rats. Two experiments were done on three groups of aged rats (30 wk) given the diets containing 20% casein, 5% casein, or 0% casein (experiment 1), and 20% casein, 20% gluten, or 20% gelatin (experiment 2) for 10 d. The aggregation in brain ribosomes declined with a decrease of quantity and quality of dietary protein except in the hippocampus. The RNA concentration (mg RNA/g protein) did not differ among the three groups varying the dietary protein in any brain regions. The results suggest that the higher quantity and quality of dietary protein improves the polysome profile in the brain of aged rats, and that the polysome profile is at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.
3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is known as a dietary carcinogen and it requires metabolic activation by cytochrome P450 (CYP) 1A subfamily to have carcinogenicity. On the other hand, our previous report demonstrated that Trp-P-1 induces apoptosis in primary cultured rat hepatocytes, but the metabolically activated Trp-P-1 added extracelluarly to hepatocytes did not induce apoptosis. In this study, we focused on the intracellular status of CYPs and investigated apoptotic events induced by Trp-P-1 using hepatocytes isolated from rats treated with three chemical inducers for CYPs. In cultured hepatocytes from rats treated with 3-methylchoranthrene, which mainly induces CYP 1A, Trp-P-1-induced apoptosis was suppressed. In the same cultures, intact Trp-P-1 was decreased and its metabolites were increased. Phenobarbital and pyridine did not affect Trp-P-1-induced apoptosis. These results suggested that evoking CYP 1A activity might interfere with apoptosis induced by Trp-P-1 in rat hepatocytes under the ex vivo system.
β-Carotene and α-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of β-carotene and reactive oxygens, and 17 lipid-radical scavenging products of α-tocopherol. The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9. None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria. Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines. So, we also measured the activity to increase 3-amino-1-methyl- 5H-pyrido[4,3-b]indole (Trp-P-2) mutagenicity. The products from β-carotene and α-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2. Thus, the products of β-carotene and α-tocopherol formed after the antioxidant actions were not genotoxic.
Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (−)-epigallocatechin gallate > gallic acid > (−)-epigallocatechin (EGC) > (−)-epicatechin gallate >ethyl gallate>> (+)-catechin=(−)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4´ position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.
Some of the food-derived tripeptides with angiotensin converting enzyme (ACE)-inhibitory activity have been reported to be hypotensive after being orally administered. The mechanism for the intestinal transport of these tripeptides was studied by using monolayer- cultured human intestinal Caco-2 cells which express many enterocyte-like functions including the peptide transporter(PepT1)-mediated transport system. Val-Pro-Pro, an ACE-inhibitory peptide from fermented milk, was used as a model tripeptide. A significant amount of intact Val-Pro-Pro was transported across the Caco-2 cell monolayer. This transport was hardly inhibited by a competitive substrate for PepT1. Since no intact Val-Pro-Pro was detected in the cells, Val-Pro-Pro apically taken by Caco-2 cells via PepT1 was likely to have been quickly hydrolyzed by intracellular peptidases, producing free Val and Pro. These findings suggest that PepT1-mediated transport was not involved in the transepithelial transport of intact Val-Pro-Pro. Paracellular diffusion is suggested to have been the main mechanism for the transport of intact Val-Pro-Pro across the Caco-2 cell monolayer.
α-Tocotrienol (α-T3) has been suggested to protect cellular membranes against free radical damage. This study was done to estimate the effect of α-T3 on free radical-induced impairment of erythrocyte deformability by comparing it to α-tocopherol (α-T). An erythrocyte suspension containing 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was forced to flow through microchannels with an equivalent diameter of 7 μm for measuring erythrocyte deformability. A higher concentration of AAPH caused a marked decrease in erythrocyte deformability with concomitant increase of membranous lipid peroxidation. Treatment of erythrocytes with α-T or α-T3 suppressed the impairment of erythrocyte deformability as well as membranous lipid peroxidation and they also increased erythrocyte deformability even in the absence of AAPH. In these cases, the protecting effect of α-T3 was significantly higher than that of α-T. We emphasize that higher incorporating activity of α-T3 into erythrocyte membranes seems to be the most important reason for higher protection against erythrocyte oxidation and impairment its deformability.
The effects of an orally administered hot-water extract (HW) from cultured mycelia of Cordyceps sinensis on the activation of macrophages and the intestinal immune system were studied in mice. The general composition of HW was 83.9% carbohydrate, 11.8% protein, 1.9% lipid and 2.4% ash, and the carbohydrates were mainly composed of glucose, mannose, galactose and arabinose (molar ratio of 1.0:0.8:0.5:0.1). HW stimulated the activation (1.7-fold of the saline control) of macrophages and IL-6 production (1.5-fold) at 2.0 g/kg/day. Analyzing the culture supernatant of Peyer's patch cells from C3H/HeJ mice that had been fed with HW at 1.0 g/kg/day for 7 days indicated that the bone marrow cells had significantly proliferated (1.9-fold). In addition, the amounts of GM-CSF and IL-6 in the culture supernatant of Peyer's patch cells at the same dose were significantly increased (1.8-fold and 2.2-fold, respectively). These results indicate that an oral administration of HW may modulate IL-6 production by the activation of macrophages, and also enhance the secretion of hematopoietic growth factors such as GM-CSF and IL-6 from Peyer's patch cells. Since such cytokines as GM-CSF and IL-6 from Peyer's patch cells act on the systemic immune system, it can be assumed that orally administered HW modulated not only the local but also systemic immune system.
A Euglena gracilis Z strain was used as a biomarker to examine the detoxifying effects of desalinated deep seawater (DDSW) and surface seawater (DSSW) on the hazardous chemical, tributyltin chloride (TBTCl). A distinct restoration effect on cell motility was observed after incubating with DDSW. The effect was largely attributed to magnesium, calcium and silicon that are involved in cell motility and morphology.
The author modified a respiratory gas analyzer to analyze the respiratory 13CO2 of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (IV) given glucose solutions containing three different amounts of 13C-labeled glucose. Expired 13CO2 derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in IV mice. The height of the peak of 13CO2 expiration was correlated with the administered 13C-glucose mass.
Compositional changes of Citrus tamurana Hort. ex Tanaka (hyuganatsu) essential oil were analyzed after storage for 1, 3, 6, and 9 weeks at −21, 5, 20, and 30°C. The total amount of oxides increased greatly. The contents of monoterpene alcohols and ketones also increased. The total amount of monoterpene hydrocarbons was unchanged up to 30°C. However, the contents of myrcene, γ-terpinene, and terpinolene decreased, while there was a considerable increase in p-cymene. Among the sesquiterpene hydrocarbons, considerable increases in the (−)-cedrene, γ-elemene, and α-humullene contents were noted in samples stored at 30°C, and the trans-β-farnesene content decreased during storage. The total content of monoterpene alcohols was increased slightly. The content of trans-carveol increased during storage. p-Cymene, trans-carveol, isopiperitone, and limonene oxide contents increased, while γ-terpinene, terpinolene, and citronellal contents decreased significantly during storage. The changes of these compounds during storage can serve as a quality index for hyuganatsu essential oil.
We found six bacteria capable of producing antifreeze protein (AFP) from Ross Island, Antarctica. Among these AFP-producing bacteria, strain No. 82 had the highest antifreeze activity and was identified as Moraxella sp. The optimum temperature and pH for the production of AFP were 5°C and 7.0, respectively. After partially purifying the AFP from the culture supernatant using 60% saturation of ammonium sulfate, only the 52-kDa protein band (100 μg/ml) which eluted from SDS-PAGE indicated antifreeze activity by the formation of hexagonal crystals. Furthermore, we confirmed that this AFP was a lipoprotein by the lipid stain test and treatment with some enzymes and that it had no ice-nucleating activity. Also, the N-terminal amino acid sequence of this AFP had high similarity with that of outer membrane proteins from Moraxella (Branhamella) catarrhalis. This is the first report of AFP-producing bacteria in Antarctica and an antifreeze lipoprotein (AFLP) from Moraxella sp.
The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58°C. It had a Km of 6.5 mg/ml and a Vmax of 435 μmol•min−1•mg−1 when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Eschelichia coli β-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.
Membrane-bound NAD(P)-independent malate dehydrogenase (EC 188.8.131.52) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.
Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from meso-erythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized meso-erythritol to L-erythrulose irreversibly with almost 100% of recovery at 37°C. L-Erythrulose was also produced efficiently by the resting cells at 37°C with 85% recovery. The enzyme responsible for meso-erythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L- erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound meso-erythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between meso-erythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0–10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.
When brain-derived neurotrophic factor (BDNF) is produced in the Escherichia coli periplasm, insoluble BDNF proteins with low biological activity and having mismatched disulfide linkages are formed. The coexpression of cysteine oxidoreductases (DsbA and DsbC) and membrane-bound enzymes (DsbB and DsbD), which play an important role in the formation of disulfide bonds in the periplasm, was investigated to improve the production of soluble and biologically active BDNF. The expression levels of Dsb proteins changed when the growth medium and the Dsb expression plasmids were changed, and the production rate of soluble BDNF was almost proportional to the expression level of DsbC protein with disulfide isomerase activity in the case of a low expression level of BDNF. The rate of soluble BDNF production with coexpression of DsbABCD was as high as 35%. These results show that coexpression of BDNF and Dsb proteins can effectively increase the production of soluble and biologically active BDNF.
We analyzed the kinetics and metabolic pathways of trichloroethylene and 1,1,1-trichloroethane degradation by the ethane-utilizing Mycobacterium sp. TA27. The apparent Vmax and Km of trichloroethylene were 9.8 nmol min−1 mg of cells−1 and 61.9 μM, respectively. The apparent Vmax and Km of 1,1,1-trichloroethane were 0.11 nmol min−1 mg of cells−1 and 3.1 μM, respectively. 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid were detected as metabolites of trichloroethylene. 2,2,2-trichloroethanol, trichloroacetic acid, and dichloroacetic acid were also detected as metabolites of 1,1,1-trichloroethane. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid derived from the degradation of 3.60 μmol trichloroethylene were 0.16 μmol (4.4%), 0.11 μmol (3.1%), 0.02 μmol (0.6%), and 0.02 μmol (0.6%), respectively. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid and dichloroacetic acid derived from the degradation of 1.73 μmol 1,1,1-trichloroethane were 1.48 μmol (85.5%), 0.22 μmol (12.7%), and 0.02 μmol (1.2%), respectively. More than 90% of theoretical total chloride was released in trichloroethylene degradation. Chloral and 2,2,2-trichloroethanol were transformed into each other, and were finally converted to trichloroacetic acid, and dichloroacetic acid. Trichloroacetic acid and dichloroacetic acid were not degraded by strain TA27.
Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.
Racemic indan derivatives have been resolved by the hydrolysis of amide bonds using Corynebacterium ammoniagenes IFO12612 to produce (S)-amine and (R)-amides. In the kinetic resolution of 1 (N-[2-(6-methoxy-indan-1-yl)ethyl]acetamide), it was possible to run the reaction to 44% conversion on a 10-g scale, obtaining (S)-amine 4 ((S)-2-(6-methoxy-indan-1-yl)ethylamine) at >99% enantiomeric excess (ee) and (R)-1 at 98% ee.
The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.