The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH₂-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH₂-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an "intramolecular chaperone, " in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane.

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This paper reports the effect of modification of lysine residues on the adsorption of ovalbumin at alumina/water interface. It has been shown that the pH dependence of the adsorption changes on acetylation of lysine. Thus at pH 7.6 acetylated ovalbumin does not show any affinity for alumina surface although unmodified protein does. It seems that although electrostatic interactions are operative, surface unfolding of proteins and surface hydrophobicity of protein also control the adsorption of ovalbumin onto alumina.

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Several strains of Corynebacterium and Brevibacterium are known for their ability to secrete large amounts of amino acids, especially L-glutamate. We focused on the mechanism of L-glutamate secretion triggered by a detergent, namely polyoxyethylenesorbitan monopalmitate (PESP). A mutant strain, AJ11060, derived from Brevibacterium lactofermentum ATCC 13869 indicates the sensitivity to PESP. A multicopy suppresser gene that compliments the sensitivity of AJ11060 to the detergent was derived from a gene library of B. lactofermentum AJ12036. A 2855-bp DNA fragment was cloned and sequenced. An open reading frame was found that coded for the rescuer gene of the sensitivity to PESP of AJ11060 and was designated dtsR. The expression of the dtsR gene in B. lactofermentum was confirmed by using anti-DtsR antibody. The deduced DtsR protein indicated significant homology with some biotin enzymes such as the β chain of propionyl-CoA carboxylase from rat (48.3%) and human (48.7%), or a 12S chain of methylmalonyl-CoA carboxyltransferase from Propionibacterium freudenreichii (43.1%).

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Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.

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We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895-898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken. The purified inhibitor migrated with the same mobility on SDS-PAGE as apo A-I, and had a molecular weight of 27, 000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.

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The metabolic fates of the carbon skeletons of leucine, lysine, and threonine were studied in growing rats on the diets containing graded levels of protein calorie percentages (10, 20, 30, and 40 PC% ) by use of either gluten or zein at 4100 kcal of metabolizable energy per kg of diets. In growth experiment for 21 days, body weight gain, food intake, and body fat increased at higher PC% in the gluten diets, but rats given zein did not maintain their initial weight even at 40 PC% . The concentration of plasma free lysine remained low with the zein diets, but plasma threonine increased at 10 and 20PC% in the gluten and zein diets, respectively. Plasma leucine increased as the protein level increased either dietary protein. More than 70% of ¹⁴C was incorporated into body protein 12h after an intraperitoneal injection of labeled lysine in all groups, but little ¹⁴CO₂ was expired in rats on the gluten and zein diets. About 79% of ¹⁴C-threonine was incorporated into body protein in rats given the gluten and zein diets at 10 PC%, but the values were gradually decreased with increasing the dietary protein levels. Some 40-50% of ¹⁴C-leucine was incorporated into the body protein in rats given the gluten diets, and the values for the zein diets were extensively decreased in the higher PC% groups where the expired ¹⁴CO₂ was inversely increased to a great extent. These results showed that, when a specific amino acid was limiting or deficient in the diet, the major portion of the labeled amino acid was utilized for body protein synthesis and little was oxidized to carbon dioxide, whereas the oxidative degradation of essential amino acid other than limiting one was increased and the efficiency of the amino acid utilization was relatively decreased.

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Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73a, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common cis-element-like sequences in phenylpropanoid biosynthesis genes.

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Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mM sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS-PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5-12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mM and 27.1 s^-1, respectively, and those for chitin pentamer were 414 μM and 83.2 s^-1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis-Menten kinetics.

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Resting cells of methanol-utilizing microorganisms isolated from soils were examined for L-serine production under conditions in which L-serine-degradation was suppressed. Strain MN43, a facultative methylotrophic bacterium identified as a Methylobacterium sp., was selected for further studies. Under the optimal conditions, 65 mg/ml L-serine was produced by this bacterium from 50 mg/ml glycine and 104 mg/ml methanol in 5 days, with a molar conversion ratio from glycine to L-serine of 93% . This production is the highest so far reported for microbes producing L-serine.

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A polyclonal antiserum against cysteine synthase (CSase, EC 4.2.99.8) isozymes (CSases 1, 2, and 3) was prepared for physiological study of the isozymes. From the results of immunotitration and double immunodiffusion analyses, the polyclonal antiserum was proved to have the same degree of reactivity toward each isozyme. The accumulation of the isozymes during germination of spinach seeds was investigated by Western blot analysis. Although CSase 1 was not detected in the cotyledons of dark-grown seedlings, it was found in the green cotyledons of seedlings that had been grown in the light. In the extract of roots, CSase 1 was not detected in any developmental stage. On the other hand, CSases 2 and 3 were detected in all developmental stages and tissues. These results indicate that the expression of CSase 1 is regulated by light, and that the regulatory mechanism of CSase 1 expression differs from those of CSase 2 and 3 expression. Western blot analysis showed that the hydrated seeds contained a new CSase band, which was different from known CSases in terms of electrophoretic mobility.

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Pseudomonas sp. strain ST-200 oxidizes cholesterol dissolved in an organic solvent overlying the medium. Major conversion products are cholest-4-en-3-one (C4EO), 6β-hydroxycholest-4-en-3-one (HCEO), and cholest-4-ene-3, 6-dione (CEDO). Productivity of each conversion product was altered by changing organic solvents used to dissolve the cholesterol. Generally, HCEO was predominant among the products. HCEO was produced even by cells grown without cholesterol and then killed with harmful organic solvent. The yield of the most oxidized product, CEDO, was improved when the cells were grown in the presence of cholesterol dissolved in a less toxic solvent, cyclooctane.

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Bacteria capable of assimilating cellulose acetate, strains SB and SC, were isolated from soil on a medium containing cellulose acetate as a carbon source, and identified as Neisseria sicca. Both strains degraded cellulose acetate membrane filters (degree of substitution, DS, mixture of 2.8 and 2.0) and textiles (DS, 2.34) in a medium containing cellulose acetate (DS, 2.34) or its oligomer, but were not able to degrade these materials in a medium containing cellobiose octaacetate. Biodegradation of cellulose acetate (DS, 1.81 and 2.34) on the basis of biochemical oxygen demand reached 51 and 40% in the culture of N sicca SB and 60 and 45% in the culture of N. sicca SC within 20 days. A decrease in the acetyl content of degraded cellulose acetate films and powder was confirmed by infrared and nuclear magnetic resonance analyses. After 10-day cultivation of N. sicca SB and SC, the number-average molecular weight of residual cellulose acetate decreased by 9 and 5%, respectively. Activities of enzymes that released acetic acid and produced reducing sugars from cellulose acetate were mainly present in the culture supernatant. Reactivity of enzymes for cellulose acetate (DS, 1.81) was higher than that for cellulose acetate (DS, 2.34).

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The pH-sensitive swelling of a xanthan/chitosan complex gel, which swelled in NaOH solutions of pH 9-12, was analyzed by the collective diffusion model developed by Tanaka and Fillmore, which is based on the diffusion of a polymer network. The time-course for swelling of the gel beads under a change in pH from 11 to 10 fitted well with the equation derived by Tanaka and Fillmore. The collective diffusion coefficient characteristic of the gel was also obtained, although the value of this coefficient was higher than that of the cooperative diffusion coefficient determined from dynamic light scattering experiments by about 4 orders of magnitude. This result indicates that the swelling rate of the gel due to pH change was not controlled by cooperative diffusion of the polymer networks.

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A model for analyzing the swelling rate of ionic gels was developed on the basis of the diffusion of a species of mobile ion. This model was applied to the analysis of pH-sensitive swelling of a xanthan/chitosan complex gel in NaOH solutions of pH 9-12, using the sodium ion as the reference mobile ion. The time-course for swelling of gel beads with a pH change from 11 to 10 was successfully described by the developed model. The values for the diffusion coefficient obtained by fitting the model to the data were of the same order as those for the diffusion coefficient of the sodium ion measured for a membrane of the complex gel. Thus, it was confirmed that the swelling rate of the gel due to pH change was mainly controlled by the diffusion of mobile ions. However, the time-course for swelling of the gel at pH values below 10 was not satisfactorily explained by the model developed, suggesting that the change in the degree of ionization during swelling also affected the swelling rate of the xanthan/chitosan complex gel.

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The groEL gene of the alkaliphilic Bacillus sp. strain C-125 was cloned in Escherichia coli and sequenced. The groEL gene encoded a polypeptide of 544 amino acids and was preceded by the incomplete groES gene, lacking its 5'-end. The sequence of the derived amino acids was 87.5% identical to that of B. subtilis, 85.4% identical to that of B. stearothemophilus, and 60.9% identical to that of E. coli. The GroEL protein was expressed in E. coli. Purified GroEL protected yeast α-glucosidase from irreversible aggregation at a high temperature and the addition of Mg-ATP was essential for reactivation of the α-glucosidase. The addition of E. coli GroES increased recovery of the enzyme activity, indicating that C-125 GroEL could function in coordination with E. coli GroES.

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The typical tastes imparted by ionic groups are salty-like of sodium chloride and umami-like of monosodium glutamate, but the relationship between the taste and chemical structure has not previously been elucidated. One of the reason for the difficulty in understanding the taste-structure relationship is the presence of the ambiguous and unfavorable tastes of neutral salts. We define the strange tastes of neutral salts (TNS) collectively in a specific category, and then the tastes due to ionic groups. Sour, salty, and umami tastes and TNS were studied for different acidic and basic groups and various combinations of both groups to elucidate the taste characteristics of the ionic groups. The results reveal that the tastes due to ionic groups have common characteristics, being different from bitter and sweet tastes.

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The syntheses and biological activities of dihydro-5, 6-dehydrokawain derivatives against plant pathogenic fungi and termites were investigated. Dihydro-5, 6-dehydrokawain was isolated by a simple method without chromatography from the leaves of Alpinia speciosa K. SCHUM. The white crystalline compound obtained was identified as dihydro-5, 6-dehydrokawain (1) by instrumental analyses. 4-Hydroxy-6-(2-phenylethyl)-2H-pyran-2-one (3) was prepared by hydrolyzing dihydro-5, 6-dehydrokawain. Three dihydro-5, 6-dehydrokawain derivatives were synthesized by reacting 3 with phosphoric agents. Among the synthesized compounds, dimethyl [6-(2-phenylethyl)-2-oxo-2H-pyran-4-yl]phosphorothionate (4) had the strongest antifungal activity of 91% at 100 ppm against Corticium rolfsii.

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The limitation of slab gel electrophoresis is in the fixed gel concentration. We devised a slab gel plate to which six different gel concentrations could be applied. Various molecular forms of Taka-amylase A (TAA) prepared by chemical modification were analyzed using this multiple lane system. Modification of Lys, Trp, and carboxyl groups of TAA gave charge isomer forms having different affinities for the substrate, soluble starch. Cross-linked oligomeric forms of TAA were resolved in monomer to tetramer forms in 6.3-12.3% gels. The affinity of TAA for various substrates was also evaluated by the multiple lanes containing different concentrations and various substrates. Moreover, the rapid and simple handling of stained gels by ethanol dehydration and subsequent lapping is described.

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Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1' positions, which is different from thermolysin.

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The pyrimidine nucleoside phosphorylase (Py-NPase) of Bacillus stearothermophilus TH 6-2 is a dimer of 46-kDa subunits and catalyzes the reversible phosphorolysis of uridine and thymidine. The gene encoding this pyrimidine nucleoside phosphorylase (pyn gene) has been cloned and sequenced from B. stearothermophilus TH 6-2. The pyn gene corresponded to an open reading frame of 1299 nucleotides that translates into a putative 433 amino acid protein with a molecular weight of 46, 271. The deduced amino terminal sequence of Py-NPase coincided with that previously found for the purified enzyme. The deduced amino acid sequence of Py-NPase shared significant similarity with those of human and Escherichia coli thymidine phosphorylases. The cloned pyn gene was overexpressed in E. coli cells to produce an active enzyme in large quantities that accounted for approximately 20% of the total protein.

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We investigated the effects of feeding various types of nicotinic acid-free, tryptophan-limiting diets on the conversion ratio of tryptophan to niacin in rats. Various tryptophan-limiting diets were made by adding zein, gelatin, glycine, threonine, methionine, or glycine+threonine+methionine to a nicotinic acid-free, 9% casein diet. When the rats were fed with the tryptophan-limiting diets, the conversion ratio of tryptophan to niacin was markedly decreased. However, the ratio recovered after the addition of tryptophan to the tryptophan-limiting diets. These results clearly prove that the conversion was lowest when the rats were fed with the tryptophan-limiting diets. Therefore, we think that the pellagragenic factor of corn is simply due to a low content of tryptophan, but the adverse effect is due to a low conversion ratio of tryptophan to niacin.

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The water-soluble polysaccharide fraction from Gracilaria verrucosa (GWS) has been reported to increase the phagocytic activity of mice [Yoshizawa et al., Nippon Shokuhin Kogyo Gakkaishi, 41, 557-560 (1994)]. In this study, the macrophage-stimulation activity of enzyme-degraded GWS (GWS-E) was investigated by intraperitoneally and orally administering GWS-E to mice. The intraperitoneal administration of GWS-E increased the number of peritoneal exudate cells (PEC), and increased the phagocytic activity and oxygen radical-secreting activity (spontaneous chemiluminescence) of PEC. This administration could also stimulate splenic macrophages (SPM), increasing radical-secreting activity. When GWS-E was administered orally, the radical-secreting activity of PEC and SPM increased. In this case of oral administration, the activity of SPM increased in a dose-dependent manner, while that of PEC had an optimum dose. These results indicate that GWS-E had macrophage-stimulation activity in vivo and would be suitable as a source for a physiologically functional food with protective and immunopotentiating activity.

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Nicardipine and nifedipine, Ca channel blockers, inhibited rat liver microsomal desaturases, though verapamil, methoxyverapamil, cinnarizine, flunarizine, and diltiazem did not. However, nicardipine and nifedipine apparently did not inhibit the fungal desaturation in Mortierella alpina 1S-4. Nicardipine inhibited rat liver microsomal Δ5 desaturase specifically (50% inhibitory concentration, 170μM), and nifedipine inhibited Δ6 desaturase specifically (78μM). The inhibition by nicardipine and nifedipine is uncompetitive, the K_i values for Δ5 and Δ6 desaturases being 62 and 44μM, respectively.

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Transfructosylated derivatives of cyclofructans (CFs) were synthesized using enzymes from Bacillus subtilis MCI-2834. The structures of the transfer products were analyzed by FBMS spectrometry and ¹³C-NMR, and HPLC with acid- and enzymatic hydrolysis products. It has been proven that these products are novel branched-chain oligosaccharides composed of cyclohexaose and fructose residues.

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In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affect the EnvZ function are discussed.

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A cryptic plasmid (pSRY1) was found in Torulaspora delbrueckii No. 3110. A plasmid vector (pSRY21) was constructed by ligating it to pPR1-RIM-C ORF with a cycloheximide resistance gene (RIM-C). The transformation efficiency of the strain No. 3110 with pSRY21 by the protoplast-PEG method was increased by 3000-fold by treatment of the protoplasts with polylysine [poly(ε-L-lysine)]. Previously we have reported a mutant of No. 3110, T1, which can neither assimilate nor accumulate trehalose. The mutant was salt- and temperature-sensitive. We cloned a DNA fragment from No. 3110, which, when introduced, complemented these mutant characters of T1. These results suggested an important role of metabolism of trehalose for the stress tolerance.

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Mepanipyrim, N-(4-methyl-6-prop-1-ynylpyrimidin-2-yl)aniline, diminished the cell surface expression of envelope glycoproteins of Newcastle disease and vesicular stomatitis viruses at concentrations where their synthesis was not profoundly affected. Intoxication by diphtheria toxin and ricin and recycling of transferrin were not affected even when cells were treated with mepanipyrim for 2 h before the addition of these probes, indicating that mepanipyrim does not act on the endocytic and recycling pathways of these proteins. Metabolic conversion of C₆-NBD-ceramide to sphingomyelin and its back-exchange to the medium was also not affected, but synthesis and back-exchange of C₆-NBD-glucosylceramide were greatly influenced, and an accumulation of LDL-derived, unesterified cholesterol was induced by the drug. These results are discussed relating to the site(s) of action of mepanipyrim.

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The antimutagenic effects of methanolic extracts of peanut hulls (MEPH) were evaluated by the Ames test. MEPH inhibited the mutagenicity of 4-nitroquinoline-N-oxide (NQNO), a direct-acting mutagen. MEPH also inhibited the mutagenicity of some indirect-acting mutagens and decreased in the order of 2-amino-3-methyl-imidazo(4, 5-f)quinoline (IQ)> aflatoxin B₁ (AFB₁)>2-amino-6-methyldipyrido(1, 2-a:3', 2'-d)imidazole (Glu-P-1)>3-amino-1, 4-dimethyl-5H-pyridol(4, 3-b)indole (Trp-P-1)> benzo(a)pyrene (B(a)P) for S. typhimurium TA98, and IQ>Trp-P-1>Glu-P-1>AFB₁>B(a)P for S. typhimurium TA100.

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A crystalline product resulted from the reduction and acetylation of the green pigment formed from the reaction of methyl caffeate and butylamine. Its structure was determined to be that of a novel benzacridine, i.e., dimethyl-7-butyl-6, 9, 10-triacetoxybenz[k, l]-acridine-1, 2-dicarboxylate.

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The effects of ascorbic acid on the deodorizing activity of polyphenols against methanethiol (CH₃SH) was investigated. The addition of ascorbic acid caused loss of the deodorizing activity of polyphenols against CH₃SH. It was assumed that ascorbic acid prevented the oxidation of polyphenols, which is the essential step for the deodorizing action against CH₃SH and so suppressed the deodorizing activity.

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The cell wall of Fusarium oxysporum was digested with commercial Bacillus pumilus chitosanase. The chitosanase produced low molecular weight heterooligosaccharides consisting of GlcN and GlcNAc from the cell wall. A main component of the digestion products was identified as 2-amino-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-D-glucopyranose. The chitosanase appeared to be more effective than Streptomyces griseus chitinase for cell wall digestion. Moreover, maltose was unexpectedly found in the digestion products, indicating that the cell wall contains α-1, 4-linked glucan chain as a polysaccharide component.

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For use as the internal standards in a quantitative analysis of natural jasmonic acid (JA) and methyl jasmonate (JAMe) by gas chromatography-mass spectrometry-selected ion monitoring, (±)-2-(2, 3-²H₂)JA and its methyl ester, (±)-2-(2, 3-²H₂)JAMe, were efficiently prepared from 2-(2-pentyl)-2-cyclopentenone through catalytic semi-deuteriogenation of acetylenic intermediates with deuterium gas in pyridine.

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Water-soluble (cacao pigment, cochineal pigment, corn pigment, betanin, carthamus yellow, and monascus pigment) and water-insoluble (gardenia yellow, laccaic acid, bixin, and curcumin) natural colorings inhibited IgE production by rat spleen lymphocytes at 10 and 1μM, respectively. Although many of those colorings only inhibited the production of IgG and IgM at high concentrations, the water-insoluble colorings enhanced IgM production even at 1μM. These results suggest that natural colorings have immunoglobulin production-regulating activity.

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Five antioxidative compounds were isolated from dichloromethane extracts of kokuto, a kind of non-centrifugal sugar, and identified by spectral analysis as syringaresinol (1), medioresinol (2), coniferyl alcohol (3), sinapyl alcohol (4), and 3-hydroxy-1-(4-hydroxy-3, 5-dimethoxyphenyl)-1-propanone (5). The antioxidative activities of 1 as determined by the ferric thiocyanate method and by the thiobarbituric acid (TBA) method were respectively superior to and similar to those of butylated hydroxyanisole (BHA). 2 and 5 had stronger activities than BHA, while 3 and 4 respectively had almost the same and weaker activities than BHA.

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A simple purification procedure for raw starch-adsorbable and -digesting amylases (RSAs) was devised. The method depended on an affinity column, which was prepared by mixing raw corn starch and Hyflo Super-Cel. RSAs were specifically adsorbed on the matrix, and eluted with a buffer containing 1% β-cyclodextrin. This column could be used to purify RSAs from Streptomyces thermo-cyaneoviolaceus and a recombinant strain of E. coli.

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The genes encoding new trehalose-producing enzymes from S. acidocaldarius ATCC33909 were cloned to analyze the distribution of these genes in Sulfolobales. Comparison of the amino acid sequences with S. solfataricus KM1 showed approximately 50% similarity. Southern analysis suggests that homologues of the trehalose-producing enzyme genes exist widely in Sulfolobales and strains in Sulfolobales were classified into three kinds of genotypes.

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Isoleucine (Ile) is a precursor for the biosynthesis of anteiso-fatty acids in rat skin, and among the four possible stereoisomers of Ile, L-Ile, and L-allo-Ile were selectively used for biosynthesis of anteiso-fatty acids. This study examined the optical rotation of anteiso-fatty acid derived from DL-Ile to ascertain its stereoconfiguration. Specific rotation of anteiso-fatty acid derived from DL-Ile favorably compared with that derived from L-Ile, suggesting the selective biosynthesis of the (S)-enantiomer of anteiso-fatty acid in rat skin.

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Perforin- and Fas-based killing pathways are two major mechanisms of cytotoxic T lymphocytes (CTL)-mediated cytotoxicity. In this paper, we have reported the identification of low molecular weight probes on CTL-mediated cytolysis. In addition to inhibitors of acidification so far reported, three other groups of compounds have been identified to block perforin-based cytolysis by the CD8⁺ CTL clone: (1) an inhibitor of actin polymerization (cytochalasin D), (2) respiratory inhibitors (antimycin A and oligomycin A), and (3) protein kinase inhibitors (calphostin C, herbimycin A, K252a, and staurosporine). Since Fas-based cytolysis by CD4⁺ CTL clone was inhibitable or rather increased by these agents, only vacuolar type H⁺-ATPase inhibitors such as concanamycin A have been shown to be highly specific probes to block perforin-based CTL-mediated cytotoxicity.

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A vacuolar type H⁺-ATPase inhibitor, concanamycin A (CMA), raised the pH and induced morphologic changes such as vacuolation in lytic granules in the CD8⁺ cytotoxic T lymphocyte clone OE4. Here we measured the pH in the lytic granules present in OE4 by immunoelectron microscopic observation using 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) as a pH probe. In control OE4, only the peripheral region, but not cores, were heavily stained with DAMP. Exposure to 100 nM CMA for 60 min raised the pH in the lytic granules from 5.7±0.2 to 6.8±0.2. Furthermore, the number of lytic granules in OE4 was estimated under phase contrast microscopy after Wright staining. About 20 lytic granules in average were detected in control cells, while CMA induced vacuolation and decreased their number by 20 to 30%.

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A new oleanane triterpene was isolated from okara fermented with Penicillium simplicissimum ATCC 90288. Its structure was established to be 7β, 15α, 24-trihydroxyolean-12-en-3, 11, 22-trione by spectroscopic techniques and an X-ray crystallographic analysis.

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Magnesium deficiency was induced in male Wistar rats by adding an excess of phosphorus and calcium to the diet (1.195g of phosphorus and 1.04g of calcium/100g of diet). Feeding of these animals with a diet containing β1→4 linked galactooligosaccharides (4'-GOS) (5g of 4'-GOS/100 g of diet) increased the apparent magnesium absorption ratios and the concentrations of magnesium in the serum and femur, and reduced accumulation of calcium in the kidney and heart. We speculate that the use of magnesium increased by feeding 4'-GOS to a limited extent prevented the lower magnesium status and the severity of calcification of the kidney and heart caused by excess dietary phosphorus and calcium.

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Transglutaminase is a calcium-dependent enzyme that catalyzes an amine incorporation and a cross-linking of proteins. Intracellular transglutaminase is induced when human promyelocytic leukemia HL-60 cells are treated with retinoic acid and human hepatoblastoma HepG2 cells, with interleukin-6. To find whether the intracellular reaction catalyzed by transglutaminase increases when the enzyme is induced in these cells, the transglutaminase-catalyzed incorportation of ¹⁴C-labeled methylamine into cellular proteins was measured. The incorporation level of the labeled methylamine into proteins of HL-60 and HepG2 cells did not increase after the transglutaminase had been induced. The presence of the calcium ionophore A23187 did not affect these results. These findings suggested that even after the enzyme induction the catalytic action of intracellular transglutaminase is maintained at a constant level in these cells by unknown regulatory mechanism(s).

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ATP sulfurylase, which catalyzes the first step on the sulfate activation, was purified to apparent homogeneity from Bacillus stearothermophilus, with ammonium sulfate fractionation and successive column chromatography. The enzyme had an apparent molecular mass of 100 kDa, consisting of two equal-sized-44 kDa subunits. The optimum pH was about 8.5-8.7. Divalent cations such as Mn²⁺, Mg²⁺, and Co²⁺ were required for its activity. Apparent K_m values for ATP and SO₄^2- were 0.045 mM and 0.2 mM, respectively. The enzyme can use deoxyadenosine 5'-triphosphate as a substrate. The enzyme was thermostable and did not show significant loss of activity for 15 min of incubation up to 70°C, suggesting a practical use for a continuous reaction.

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A novel mannanase that has a strict specificity for the α-1, 3-mannosidic linkage has been isolated and characterized. The enzyme was purified to homogeneity from the cultural supernatant of a soil bacterium, Flavobacterium sp. AS-9 isolated by enrichment culture on Auricularia mannan of which the structure was an α-1, 3-linked D-mannan main chain branched with short segments of xylose or glucuronic acid. Purified mannanase was optimally active between pH 6 and 8. The apparent molecular mass of the enzyme was 94 kDa by SDS gel electrophoresis under reducing conditions and 100 kDa by gel filtration HPLC. The enzyme only hydrolyzed α-1, 3-D-mannan with endo-type action to produce α-1, 3-mannooligosaccharides of various sizes. The smallest product was α-1, 3-mannobiose.

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A new cysteine protease inhibitor, named kojistatin A, was found in the extract of a solid culture of Aspergillus oryzae ATCC 20386. The structure of kojistatin A was determined on the basis of chemical and spectroscopic evidence. Kojistatin A specifically inhibited cysteine proteases such as papain, ficin, and bromelain.

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