Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inducible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.
The absolute configuration of the chiral center at the C15 position of a novel branched-chain fatty acid derived from a new ceramide isolated from the epiphytic dinoflagellate Coolia monotis was determined to be of R from by reversed-phase HPLC after cleavage to 12-methylpentadecanoic acid and subsequent conversion with the chiral fluorescent reagent, (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol.
A cell suspension culture of table beet (Beta vulgaris L.) was established for efficient betacyanin production from violet callus induced from the hypocotyls of aseptic seedlings. This suspension culture produced large amounts of betacyanins. The betacyanin content increased with increasing cell growth during the log phase. Reducing the total nitrogen concentration (30 mM) and modifying the ratio of ammonium to nitrate (1:14) resulted in an increased betacyanin content. Supplementation of Fe2+ to the LS medium also promoted betacyanin production. The maximal betacyanin yield was achieved with a 2 mM Fe2+ concentration. Combining these conditions, we established a revised LS medium to improve betacyanin productivity (250 mg/l for a 14-day culture).
2-C-Methyl-D-erythritol (A) and 2-C-methyl-L-threitol (B) were respectively synthesized from D-glucose and D-galactose. The 2-C-methyl-1,2,3,4-butanetetrol compound (C) recently isolated from Phlox sublata L was confirmed to be A by comparing the CD and 1H-NMR spoectra of its tri-O-benzoate with those of A and B.
The oviposition response of the Rutaceae-feeding swallowtail butterfly, Papilio bianor, was induced by a methanolic extract from leaves of its major host, Orixa japonica. Several components were responsible for this oviposition response. One of the stimulants was isolated and identified as (-)-2-C-methyl-D-erythrono-1,4-lactone. The compound was inactive alone, but elicited oviposition behavior when mixed with other fractions.
D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.
5-Oxa-7-epi-jasmonic acid and 5-oxa-jasmonic acid, which are stereochemically restricted lactone-type analogues of jasmonic acids, were synthesized via three-component coupling of 2(5H)-furanone, tert-butyl acetate and 1-bromo-2-pentyne. After acidic deprotection of the tert-butyl esters, the (Z)-olefin was introduced by catalytic partial reduction with the Lindlar catalyst to give the desired analogues.
We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.
Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.22.214.171.124) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction. A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2′-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2′-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.
The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9~5GlcNAc1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9~5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1- 6(Man1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.
The structural analysis of monoclonal antibodies (mAbs) against the α subunit of the high affinity IgE receptor (FcεRIα) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcεRIα and to analyzing the various biological effects induced by anti-FcεRIα autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcεRIα mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcεRIα containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcεΡΙ (CHO/αβγ), and these inhibited the binding of IgE to CHO/αβγ cells.
Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SATΔC10-ΔC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATΔC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATΔC10 was inactivated on cooling to 0°C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-Ile mutant SAT as well as SATΔC14, SATΔC20, and SATΔC25 were stable. Since SATΔC10, SATΔC14, and SATΔC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATΔC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.
Agonistic anti-human Fas antibodies that can induce apoptosis are thought to have therapeutic effects for various diseases resulting from an abnormality of the Fas/FasL system. However, some anti-Fas antibodies show toxicity, and it is difficult to investigate their therapeutic and toxicological effect using animals because of their species specificity. We previously obtained a murine anti-human Fas mAb, HFE7A. HFE7A reacted with both human and murine Fas, and mitigated lymphadenopathy without any sign of hepatotoxicity in MRLgld/gld mice. It is suggested that humanized HFE7A would be a therapeutic treatment for various diseases resulting from an abnormality of the Fas/FasL system. Here we isolated the cDNAs that code for the heavy and light chains of HFE7A and identified the corresponding nucleotide sequences. The recombinant HFE7A was indistinguishable in binding and apoptosis-inducing activity to that from a hybridoma cell line. These data provide essential information for the humanization and clinical application of the humanized HFE7A.
Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c). To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c. Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79°C and 90°C, respectively. Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate. E3d lost FAD, and inactive E3d polypeptides were aggregated. The sulfonate bound to the aggregate became notably fluorescent. The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex. Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.
In this study, we addressed the question of which genes are transcriptionally co-regulated upon proliferation of the endoplasmic reticulum, as induced by an experimental overexpression of cytochrome P450Cm2 (CYP52A4) in Saccharomyces cerevisiae. Using the mRNA differential display technique, six genes were found to be up-regulated: ASN2, MDJ1, YLR194c, YNL208w, YER175, and YGL121c. Genes coding for Dur1.2p, Dal2p, and Sps19p were down-regulated. Two strongly induced genes, which were found to accommodate the peroxisome box (YLR194c) and a 10-bp consensus sequence of genes involved in lipid metabolism (YNL208w) in their promoter regions, were further analyzed with respect to the course of induction, the necessity of the P450 membrane anchor for induction, and the effects of gene disruption on P450Cm2 overexpression. We found that both genes are not essential to overproduce P450Cm2, but their induction was dependent on P450Cm2 membrane integration.
The Saccharomyces cerevisiaeVIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate. Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity. During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability. JS30 excreted cell surface proteins in the medium without any indication of cell lysis. Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene. These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity.
An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (Δzds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.
The complete nucleotide sequences of a cDNA (RSP1) that encodes a subtilisin-like serine protease (subtilase) of rice (Oryza sativa L.) and a gene (ASP48) for Arabidopsis subtilase were analyzed. The RSP1 cDNA and ASP48 DNA encoded 736- and 757-residue pre-pro-polypeptides including a signal peptide with molecular masses of 78,668 Da and 79,414 Da, respectively. RSP1 is the first known serine protease in rice, and ASP48 is a gene for ara12 cDNA. Sequence comparison and phylogenetic analysis showed that RSP1 is distantly related to all other plant subtilases and ASP48 is closely related to a tomato subtilase, SBT1. The ASP48 gene was found to lack introns. The Arabidopsis subtilase gene appears to consist of a small gene family. The RSP1 was found to be expressed in seed and shoots of seedlings while ASP48 transcripts was found to be accumulated in immature silique and flowers, indicating that both RSP1 and ASP48 are organ-specific and may be involved in the specific proteolytic events that occur during organ development.
The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.
The spike G protein of bacteriophage φX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the φX174-sensitive strains, and did not bind to LPSs of the φX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.
We applied ‘metal switch’ experiments to the S3 site residues, Ser102 and Gly131, of aqualysin I, a subtilisin-type serine protease. We showed that two histidines introduced at these positions did take part in histidine-metal-histidine bridge formation, and metal ions inhibited the protease activities. These results indicate that two histidines are near each other, and both side chains are metal-accessible. This is the first report on application of the metal-switch technique to a subtilisin-related enzyme.
Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, σ38, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of σ38 protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA (SerU), called, supD. Analysis of the amino acid sequence of a σ38 preparation from WC196 showed that the 33th residue of σ38 is a serine residue. The ΔrpoS ΔcadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of σ38, expressed a significant amount of Ldc and accumulated a large amount of σ38. However, the ΔrpoS ΔcadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither σ38 nor Ldc significantly.
A gene (aman6) encoding endo-1,6-α-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1767 base pairs long and encoded a mature 1,6-α-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-α-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-α-D-mannanase from Bacillus circulans TN-31.
Germinated barley foodstuff (GBF) administration has been previously reported to suppress dextran sulfate sodium (DSS)-induced experimental colitis. In this study, we investigated the roles of the intestinal microflora and short chain fatty acids (SCFAs) following administration of GBF in DSS-induced rat colitis. Sprague-Dawley rats were fed 3% (w/w of diet) DSS in GBF-diets for 5 days. The control rats were fed 3% DSS in cellulose-diets for 5 days. The administration of GBF effectively prevented bloody diarrhea and mucosal damage as compared to control rats. GBF significantly elevated fecal acetic acid and n-butyric acid levels. GBF tended to increase the number of eubacteria and that of bifidobacteria as compared to control rats. In addition, the number of enterobacteriaceae, the total number of aerobes and bacteroidaseae, were significantly lower in rats fed GBF than in the control group. It is suggested that the therapeutic effects of GBF for DSS-induced colitis depend mainly on increased SCFAs, which are accompanied by changes of composition of intestinal bacteria.
It has been found that α-D-glucosylglycerol (GG) is contained in such traditional Japanese foods brewed by using koji as sake, miso and mirin, and that GG is formed by transglucosylation to glycerol that is produced by yeast with α-glucosidase (EC 126.96.36.199) from koji in the sake mash. GG has also been found to consist of three components, 2-O-α-D-glucosylglycerol (GG-II), (2R)-1-O-α-D-glucosylglycerol (R-GG-I) and (2S)-1-O-α-D-glucosylglycerol (S-GG-I). GG was synthesized from a mixture of maltose and glycerol by the batch method, using α-glucosidase (transglucosidase L-AMANO). α-Glucosidase seemed to be so stable that the amount of GG increased about 5-fold compared with that in the first reaction by the daily addition of maltose for 10 d. Syrupy GG obtained was found to have the following characteristics: about 0.55-fold sweetness compared with sucrose, high thermo-stability, low heat-colorability, low Maillard reactivity, low hygroscopicity, high water-holding capacity, non-cariogenicity and low digestibility.
The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.
Lactobacillus delbrueckii subsp. bulgaricus 2038 was examined for its activity to prevent the oxidation of the erythrocyte membrane in vitro, and the oxidation of LDL in vivo. Strain 2038 produced radical scavengers that reacted with 1,1-diphenyl-2-picrylhydrazl (DPPH) during cultivation. Moreover, the ethereal extract from the supernatant of the culture prevented the oxidation of the erythrocyte membrane in vitro. As an in vivo study, male F344 rats were fed on diets containing 20% fresh soybean oil (or 13% oxidized oil and 7% fresh oil) with 10% freeze-dried powder of the 2038 culture (or with skim milk powder) for 4 weeks. The level of thiobarbituric acid-reactive substances was lower in the low-density lipoproteins (per milligram of cholesterol) from rats fed on the oxidized oil with freeze-dried powder of the 2038 culture than without it. The level of vitamin E in the plasma was higher in the rats fed on the oxidized oil with the freeze-dried powder than without it.
Feeding studies on rice genetically modified with soybean glycinin were performed on rats for four weeks. The rats were divided into three groups, each being fed on (I) only a commercial diet, (II) this diet plus control rice and (III) this diet plus rice genetically modified with glycinin. The rats were fed with 10 g/kg-weight of rice every day by oral administration. During the test period, the rats in every group grew well without marked differences in appearance, food intake, body weight, or cumulative body weight gain. There were also no significant differences in the blood count, blood composition or internal organ weights among the rats. Necropsy at the end of the experiment indicated neither pathological symptoms nor histopathological abnormalities in the liver and kidney. Judging from these results, the rice genetically modified with glycinin is considered to have been essentially the same in nutritional and biochemical characteristics as the control rice.
The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of ~200 kDa, ~67 kDa and ~60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the ~67 kDa component and the other had the ~200 kDa and ~60 kDa components. One of them (~67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the ~200 kDa and ~60 kDa components were presumed to be glycoproteins. Bovine gamma globulin (BGG: M.W. ~160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the ~200 kDa and ~60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the ~200 kDa, ~67 kDa and ~60 kDa components in the beef extract had strong allergenicity: ~67 kDa was BSA, and ~200 kDa and ~60 kDa were presumably aggregated BGG and it’s heavy chain, respectively.
The in vitro antioxidative activities of various kinds of vinegar were investigated by using a linoleic acid autoxidation model detected by the thiobarbituric acid (TBA) method and the 1,1-diphenyl-2-picrylhydrazyl radical system. An ethyl acetate extract of Kurosu (EK), a vinegar made from unpolished rice, exhibited the highest antioxidative activity in both systems. EK (5 mg) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema formation (14%) and myeloperoxidase activity (52%, P<0.01) in female ICR mouse skin. Furthermore, EK significantly suppressed double TPA application-induced H2O2 generation (53%, P<0.01) and lipid peroxidation determined by the TBA-reacting substance level (95%, P<0.01). In a two-stage carcinogenesis experiment with dimethylbenz[a]anthracene/TPA, EK significantly reduced the number of tumors per mouse by 36% (P<0.05) at 15 weeks after promotion. These results suggest that the antitumor-promoting effect may be partially due to the antioxidative properties of EK such as the decomposition of free radicals and interference with free radical-generating leukocytes.
A method for an enzymatic assay of histamine by using histamine oxidase from Arthrobacter globiformis in combination with amperometric determination of H2O2 is described. Histamine could be quantified at a level as low as 10-7 M. The assay is adaptable to determine histamine in food samples including tuna fish with good sensitivity and selectivity.
The tyrosinase-inhibitory activity of 15 kinds of Bangladeshi medicinal plants was evaluated. Methanol extracts were prepared for screening tests, and other kinds of extracts were also studied for those with high activity. Swertia chirata, Piper nigrum, Glycyrrhiza glabra, Piper longam and Ocimum americanum were screened as highly inhibiting samples. Methanol was found to be the most efficient solvent for extracting the active compounds. The 50% tyrosinase-inhibitory concentration of the Glycyrrhiza glabra methanol extract was 21.2 μg/ml.
A panel of tasters has found that the N-trifluoroacetyl derivative of aspartame is five times less sweet than the parent compound, contrary to the tenet in the literature, but consistent with sweet receptor models which require this nitrogen to exist in protonated form.
Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5%) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.
A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20% (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20% ethanol (6.25 ml each), with about 65% of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70% of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.
We previously demonstrated that 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induced apoptosis in primary cultured rat hepatocytes. In this study, we investigated apoptotic biomarkers in rat liver after perfusion with 30 μM Trp-P-1 as preliminary experiments for in vivo study. Induction of c-Myc and p53 protein and the activities of caspase-3, -6, and -8 were detected in Trp-P-1-perfused liver. In addition, Trp-P-1 modulated the DNA binding activity of the apoptosis-related transcription factors, NF-κΒ and AP-1. These results imply a possibility that Trp-P-1 would induce apoptosis in vivo.
The production of a highly branched β-1,3-glucan by Aureobasidium pullulans K-1 in Czapek’s medium has been found to be stimulated by ascorbic acid. When the culture supernatant, after removal of polysaccharide from the culture filtrate by ethanol precipitation, was concentrated, then added to a new medium and this strain was cultured in the medium, the polysaccharide production was stimulated the same as when L-ascorbic acid was added to the medium. The stimulating substance was partially purified from the supernatant, and was found to be oxalic acid; 0.03% oxalic acid was the most effective concentration for the stimulation of polysaccharide production. The stimulating substance, oxalic acid, was proved to be derived from ascorbic acid added to a medium in an experiment using L-[1-14C]ascorbic acid. We suggest that oxalic acid generated from the metabolism of ascorbic acid in cells of Aureobasidium pullulans K-1 participated in the stimulation of the polysaccharide production by ascorbic acid.
Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.
Degradation of 2,2-Bis(4-hydroxyphenyl)propane (bisphenol A, BPA), an endocrine-disturbing chemical, by the growing mycelia of the white-rot basidiomycete, Pleurotus ostreatus, was examined. About 80% of BPA initially present decreased in 12 days of culture with this fungus. By in vitro experiments using the lignin-degrading enzyme manganese peroxidase (MnP), BPA was metabolized to phenol, 4-isopropenylphenol, 4-isopropylphenol, and hexestrol. The degradation products of BPA were assumed to be formed by the one-electron oxidation of the substrate.
Primary rat hepatocytes form discontinuous monolayers even at their maximum density. Here, we show that staurosporine promotes attachment and spreading of hepatocytes onto culture substrates, so that hepatocytes form a close, continuous monolayer. This treatment did not attenuate major hepatic functions. Therefore, this technique is promising for making seamless cell-sheet structures, which will be applicable for cell-polarity experiments or artificial liver construction.
The 23 quaternary and tertiary protoberberines related to berberine were tested for in vitro and/or in vivo fungicidal and herbicidal activities. Among the compounds tested, there was some activity observed with some of only the protoberberinium salts, but not sufficiently strong or broad spectrum for agrochemical use. From the structure-activity point of view, some features can be pointed out.