The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers~oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2α→7, 4α→8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.
A new nematicidal alkaloid, peniprequinolone (1), together with the known alkaloids penigequinolones A and B (2a, 2b), 3-methoxy-4-hydroxy-4-(4′-methoxyphenyl)quinolinone (3), and 3-methoxy-4,6-dihydroxy-4-(4′-methoxyphenyl)quinolinone (4), were isolated from Penicillium cf. simplicissimum (Oudemans) Thom. Cyclopenin (5) and a compound (6a/6b) structurally related to cyclopenin also were isolated from the fungus, and their structures were established by spectroscopic analysis. The biological activities of 1, 2, 3, 4, and 5 were examined by a bioassay with root-lesion nematodes.
Two new anthocyanins were isolated from purple pods of pea (Pisum spp.). Their structures were identified as delphinidin 3-xylosylgalactoside-5-acetylglucoside and its deacetylated derivative by the usual chemical degradation methods and by spectroscopic methods such as UV-VIS, MS and NMR. Both pigments showed moderate stability and antioxidative activity in a neutral aqueous solution.
The effect of heat stress (38°C) on the content of DL-β-(3,4-dihydroxyphenyl)alanine (DOPA), dopamine, tyramine, octopamine, and their precursor Tyr was studied in adults of two lines of Drosophila virilis contrasting in their stress response. In individuals of line 101 responding to stress by a hormonal stress reaction, the contents of DOPA, dopamine, octopamine, and Tyr were lower than those of line 147 that did not respond to the stress. However, heat stress caused an increase in the contents of DOPA, dopamine, octopamine, and Tyr in line 101, whereas the equivalent titers in line 147 remain unchanged.
A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring α-ionylideneacetic acid from Cercospora rosicola and γ-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of [1, 2-13C2]-α and γ-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1′ and 4′. Enzymatic isomerization from the γ to α-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.
When long-chain unsaturated fatty acids such as oleic, linoleic, and linolenic acid were incubated with crude enzymes from the marine green alga Ulva pertusa, the corresponding (R)-2-hydroperoxy acids were formed with a high enantiomeric excess (>99%).
A highly potent attachment-inhibitor, polygodial, was isolated from a hexane extract of the leaves of Tasmannia lanceolata. The attachment-inhibiting activity of polygodial against the blue mussel was increased 4-fold when used in combination with sorbic acid, anethole, and indole.
The stereochemically restricted bicyclic analogue of 7-epi-jasmonic acid was synthesized from a known bicyclo[3.3.0]octane derivative. The enol triflate derived from the bicyclic compound was subjected to palladium-catalyzed coupling with allyltributyltin to give the desired carbon skeleton. Selective catalytic hydrogenation and subsequent acidic hydrolysis gave a new bicyclic analogue of 7-epi-jasmonic acid. The ACC conjugate of the bicyclic analogue was also synthesized. This ACC conjugate exhibited only slightly weaker potato cell expansion-inducing activity than that of the JA standard.
All of the four possible stereoisomers of 5,9-dimethylpentadecane, the major sex pheromone component of the coffee leaf miner moth (Perileucoptera coffeella), were synthesized by using the methyl esters of (S)- and (R)-3-hydroxy-2-methylpropanoic acid as chiral sources for the purpose of determining the stereochemistry of the pheromone.
The α-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The α-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the α-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the α-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40°C, respectively. The enzyme's half-life at 50°C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid [2-O-α- (4-O-methyl-α-D-glucopyranosyluronic)-D-xylobiose]. The α-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when α-glucuronidase was added to a mixture of endoxylanase and β-xylosidase.
Secondary structure of α-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. α-Chymotrypsin formed β-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, α-chymotrypsin took on a native-like structure, which gradually changed to β conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, α-chymotrypsin tended to form stable β-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to α-helix structure with substantial loss in catalytic activity was observed when α-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, α-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of β-sheet structure.
To elucidate the role of Elf-1 in FcεRI α chain expression, rat Elf-1 cDNAs were isolated and characterized. The rat Elf-1 cDNA of 2744 bp contained an open reading frame of 1848 bp. In addition to the full length rat Elf-1 cDNA (named type 1), two splice isoforms were isolated. One of the two isoforms lacked the amino acid residues from 85th to 120th (type 2), and the other from 85th to 175th (type 3). Similar isoforms were also observed in human tissue. Overexpression of rat Elf-1 (type 1) using a transient coexpression system inhibited of the α chain promoter activity. The inhibition activity was different between the isoforms; the inhibition activity of type 2 was lower than that of type 1, and type 3 did not have an inhibitory effect. This observation suggested that each Elf-1 isoform played a different role in the gene expression under its control.
Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase.
Staphylococcus aureus P83 has Panton-Valentine leukocidin (PVL)-like genes, lukM and lukF-PV. Here, lukM and lukF-PV genes were found on the genome of a prophage, which was designated as φPV83-pro. The precise genome size was 45,636 bp with att core sequences of 10 base pairs. Sixty-four ORFs were identified on the φPV83-pro genome, including two extra operons, lukM-lukF-PV and orfs63-64. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. The most striking feature of the φPV83-pro genome was a constituent of at least 4 regions from φ11, φPVL, and other phages, i.e., (i) att sites identical with those of φ11, (ii) a cos sequence and the genes encoding packaging and head proteins of φPVL (occupied half region of φPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40% of φPV83-pro). Furthermore, two insertion sequences, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. φPV83-pro was not induced as phage particles from S. aureus P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into the attL site was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83.
Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 106 cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T50, was significantly decreased (T50<-5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).
After exposure to thermal stress or a control temperature, the relative abundance of ecdysone (E) and 20-hydroxyecdysone (20E) was measured in a wild-type line of Drosophila virilis (101) that is stress responsive and in a mutant line (147) that is not stress responsive. In line 101, the 20E content was higher and E content lower in females than in males. The abundance of E and 20E in females of line 147 was significantly higher than that in females of line 101. Females of line 101 were found to respond to 60 min of heat stress (38°C) by an increase in the abundance of both E and 20E, while in males of this line the amount of 20E increased and that of E declined. A role of the ecdysteroids in the control of reproduction of D. virilis under stress is discussed.
We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at -20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10-10, 4.35×10-9, 8.98×10-12, and 3.05×10-11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.
We cloned cDNA of three variants of BtR175, a putative Bombyx mori receptor for Bacillus thuringiensis Cry1Aa δ-endotoxin by PCR. These variants were likely to be allelic to BtR175. cDNA of BtR175b, the most distant variant from BtR175, was introduced into mammalian cells. BtR175b protein was expressed in the plasma membrane of the cells and showed binding activity to Cry1Aa.
We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.
To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.
Hen egg white (HEW) lysozyme was correctly processed and efficiently secreted from an alternative yeast, Kluyveromyces lactis. We constructed secretion vectors using PHO5, PGK, and LAC4 promoters, and found that the highest secretion was obtained under the direction of the PGK promoter in non-selective rich medium. K. lactis secreted HEW lysozyme with two-fold higher efficiency than S. cerevisiae, estimated by using a K. lactis-S. cerevisiae shuttle vector.
Heterocapsa circularisquama (Dinophyceae), a noxious red tide dinoflagellate, is known to have a specifically lethal effect on shellfish, especially bivalves such as pearl oyster (Pinctada fucata), but no detrimental effects of this alga on fishes have not been observed so far. In this study, we found that H. circularisquama was toxic to a microzooplankton, a rotifer (Brachionus plicatilis) in a cell concentration-dependent manner, while the cultured supernatant or ultrasonic ruptured H. circularisquama had no significant toxic effect on the rotifer. Since no such toxic effects on the rotifer were observed in Chattonella marina, Heterosigma akashiwo, or Cochlodinium polykrikoides, other species of harmful red tide plankton, H. circularisquama may have a strictly specific toxic mechanism against the rotifer as well as bivalves.
Nitrite-oxidizing enzyme I (NiOx I) was purified from a heterotrophic bacterium, Bacillus badius I-73. The enzyme was a homotetramer of a heme-containing peptide, and was similar to catalases from various sources in its N-terminal amino acid sequence. The purified enzyme also catalyzed H2O2 degradation. The nitrite oxidation reaction required ascorbic acid and oxygen. Successive H2O2 feeding could be substituted for ascorbic acid. These indicated that NiOx I is a catalase and nitrite was oxidized by a peroxidase-like reaction.
It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 [6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol]. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.
Staphylococcal bi-component cytotoxins, leukocidin (Luk), Panton-Valentine leukocidin (PVL), and γ-hemolysin (Hlg) consist of LukF and LukS, LukF-PV and LukS-PV, and LukF and Hlg2, respectively, and Luk and Hlg share LukF. LukF-PV can not substitute for LukF for Hlg, despite 73% identity in amino acid sequence and close similarity in the 3-dimensional structure between them. Here, we demonstrated that the absence of hemolytic activity of LukF-PV in cooperation with Hlg2 is due to the failure of the binding of LukF-PV to human erythrocytes. We identified Y72 residue at the bottom of rim domain in LukF as the crucial residue for its binding, which is a prerequisite to the subsequent binding of Hlg2 to human erythrocytes. The data obtained showed that a mutant of LukF-PV in which T71 residue was replaced by the corresponding residue of LukF, Y72, endowed LukF-PV with the binding capability to human erythrocytes which was accompanied by its hemolytic activity in the presence of Hlg2.
The release of volatile compounds from a cream style dressing, which consisted of a thickening agent dispersed in the water phase of an oil in water (o/w) type of emulsion, was studied by the purge-and-trap (PT), dynamic head space mastication (DHM) and dynamic headspace (DH) model systems for diacetyl and 2-heptanone as two volatile compounds. Big differences were detected in the quantity of volatiles released by the three models for both diacetyl and 2-heptanone: PT released the most, followed by DHM and DH. Nitrogen gas bubbling in PT and plunger up-and-down motion in DHM mimic mouth movements and promoted volatile release more than DH. The quantity of volatiles released depended on the nitrogen gas flow rate and isolation period with both the PT and the DHM model. Static headspace measurements indicated that no interaction occurred between the volatiles and the dispersion thickening agent, nor between the volatiles and protein of saliva.
Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 μM of either linoleic acid (LA, 18:2n-6), γ-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 μM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.
Crude dietary fiber samples were prepared from beet, cabbage, Japanese radish, onion and mung bean sprouts (BF, CF, RF, OF and MF, respectively). These samples contained total dietary fiber at the levels of 814, 699, 760, 693 and 666 g/kg, respectively. To examine the effect of these dietary fiber sources on the plasma cholesterol concentration, male Sprague-Dawley rats were fed on a fiber-free (FF) diet or on an FF diet supplemented with 5% or 10% dietary fiber. Dietary fiber extracted from vegetables, wood cellulose (CL), pectin (PE) and guar gum (GG) were used as the fiber sources. Compared with the rats fed on the FF diet, a significant reduction in the plasma cholesterol concentration was observed in the rats fed on BF, CF, RF, MF, PE or GG after a 21-d feeding period. Cecal acetate, n-butyrate and total short-chain fatty acids were significantly higher in the rats fed on these dietary fibers, except for CF, than in those fed on the FF diet. A negative correlation was apparent between the total dietary fiber content, hemicellulose content and pectin content of each dietary fiber source and the plasma cholesterol concentration. These results suggest that some vegetable fibers exert a plasma cholesterol-lowering effect through cecal fermentation of these fibers.
The aqueous fraction of Fushimi sweet pepper increased the repair effect of the solvent control against UV-induced cyclobutane pyrimidine dimers in human fibroblast to 150%, but ordinary sweet pepper did not have a statistically significant effect. When Fushimi sweet pepper was boiled, the activity of the aqueous fraction was elevated to 209% of the control (p<0.05), while that of the grilled state was decreased to 125% of the control. The repair activity of a dialyzate (MW<12,000) of the aqueous fraction from Fushimi sweet pepper showed 191% of the control (p<0.05). The dialyzate was contained 1.9% in the weight of the fresh fruit body of Fushimi sweet pepper, and the activity can be stable in its boiling state, and it might be therefore considered to be the worthy source for expecting the DNA repair activity in human diet.
Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTB5 productivity were observed in the PEC, in response to the treatment with 5 μM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.
The effect on genetically obese mice of a milk whey protein isolate (WPI) and soy protein isolate (SPI) and their hydrolysates (WPI-H, SPI-H) on the rate of body fat disappearance was investigated. Male yellow KK mice were made obese by feeding with a high-fat diet containing 30% fat from 6 to 10 weeks of age. They were then fed with an energy-restricted low fat (5.0%) and high protein (35% WPI, WPI-H, SPI or SPI-H) diet for 2 weeks at the 60% level of energy intake by mice on laboratory feed. During the weight reduction period, the body weight of the WPI, WPI-H, SPI and SPI-H groups changed by -9.1, -9.1, -10.0 and -11.1 g/14 days, respectively, the reduction being significantly lower in the SPI-H group than in the WPI and WPI-H groups. The plasma total cholesterol level was significantly lower with the SPI diet, and the plasma glucose level was lower with the SPI and SPI-H diets than with the WPI and WPI-H diets. Although the body protein content was comparable in all the groups, the body fat content was significantly lower with the SPI diet than with the WPI diet, and was also significantly lower with the SPI-H diet than with the WPI and WPI-H diets. The weight of the perirenal fat pads was significantly lower with the SPI-H diet than with the WPI and WPI-H diets. These results indicate that SPI and SPI-H are suitable protein sources in an energy-restricted diet for treating obesity.
A novel method is proposed to produce hypoallergenic wheat flour suitable for patients allergic to wheat. Wheat flour was mixed with a cellulase solution, and the mixture was incubated at 50°C for 1 h to hydrolyze the carbohydrate allergens. The hydrolysate was further incubated with actinase at 40°C for 1 h while gently stirring to decompose the proteinaceous allergens. The product was evaluated for its allergenicity by an enzyme-linked immunosorbent assay, the results of which suggested negative allergenicity in most cases. The product changed to a batter state that was difficult to process by the usual methods. Gelatinization of the starch in the product and the addition of a surfactant were beneficial for food processing.
The stability and bioavailability of the major antioxidants in garland (Chrysanthemum coronarium L.), chlorogenic acid, 3,5-dicaffeoylquinic acid and 4- succinyl-3,5-dicaffeoylquinic acid, were investigated together with caffeic acid. These compounds were stable in artificial digestive juice, but more than 90% of them disappeared from plasma within 30 min after intravenous injection into rats. When they were orally administered, only caffeic acid could be detected.
Nine healthy volunteers drank fermented milk containing 4×1010 live cells of Lactobacillus casei strain Shirota daily for 3 weeks, and their NK activity and other immunological functions were measured. NK activity significantly increased (p<0.01) 3 weeks after the start of intake and remained elevated for the next 3 weeks. The effect was particularly prominent in low-NK-individuals.
The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B12 compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B12 compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B12, but not to those of vitamin B12 analogues inactive for humans.
Enterostatin (VPDPR), having anoretic and hypocholesterolemic activities, and its homologue LPYPR, a hypocholesterolemic peptide found in the glycinin A5A4B3 subunit, were introduced into the corresponding site (TNGPQ) of the proglycinin A1aB1b subunit by site-directed mutagenesis. Modified proglycinins were expressed in E. coli and recovered from the insoluble fraction. VPDPR and LPYPR were released by the action of chymotrypsin and trypsin as expected. The overall yields of purified VPDPR and LPYPR were 40% and 62%, respectively.
The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.
Pyridoxine-charged Schizosaccharomyces pombe released pyridoxine rapidly at 30°C: very low amounts of three other B6 vitamers were also released. The rate of efflux was temperature-dependent. The initial rate of efflux was dependent on the concentration of pyridoxine in the cells: the rate was almost zero at lower than 0.02 mM and became saturated at higher than 0.2 mM. Na+, sodium azide, and dinitrophenol increased the rate in both the presence and absence of D-glucose. Mg++, thiamine, and menadione inhibited the efflux. The intracellular concentration of ATP did not significantly affect the efflux rate. The system may be dependent on a membrane potential of the yeast cells. It was found that the fission yeast cells have a gate or carrier system for efflux of pyridoxine, which was distinct from that in Saccharomyces cerevisiae.
Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer- Villiger monooxygenases.
An alkaliphilic Dietzia sp., strain GS-1, which degraded disodium terephthalate (DT), was isolated from soil. Strain GS-1 degraded 19.3 mM of DT in 168 h at pH 10. The maximum degradation velocity was 0.46 mM/h. The resting cells efficiently degraded 28.7 mM of DT in 51 h at 28°C and pH 10. The degradation velocity was 0.41 mM/(h g-wet cell).
The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.