Tomatine is a steroidal glycoalkaloid in tomato plants (Lycopersicon esculentum) and other Lycopersicon and Solanum Species. Tomatine is used as an indicator to evaluate the safety of transgenic tomatoes by FDA in U.S.A. We have developed a facile and rapid method for absorptiometric measurement of the tomatine content. This method was used to measure the tomatine content of fruits of a transgenic tomato cultivar (Lycopersicon esculentum) which contained an antisense polygalacturonase gene (anti-PG). We found that the tomatine content in the fruits of transgenic and non-transgenic tomatoes was very similar. The data were also compared with those of other tomato cultivars, “KAGOME 77” (L. esculentum) and “KAGOME 88” (L. esculentum).
Two novel oxazolomycin isomers, oxazolomycins B (2) and C (3), were isolated from the fermentation broth of an oxazolomycin-producing strain, Streptomyces albus JA3453. Both compounds are geometrical isomers of oxazolomycin (1), the configurations of their triene moieties being (4′E, 6′E, 8′E) (2) and (4′Z, 6′E, 8′E) (3) while that of oxazolomycin (1) is (4′Z, 6′Z, 8′E). Compounds 2 and 3 exhibited potent inhibitory activity against crown gall formation with the same MIC (0.8 μg/disk) as oxazolomycin. Compounds 2 and 3 showed no antibacterial activity against Agrobacterium tumefaciens, in contrast to oxazolomycin which has specific anti-A. tumefaciens activity.
A facile synthetic route is described to cyanogenic glycosides (R)-prunasin, linamarin and (S)-heterodendrin from O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)trichloroace- timidate and the corresponding α-hydroxyamides by a 3-step reaction of glycosylation, cyanohydrin formation by dehydration of carboxamides, and deprotection.
The root bark of Melia toosendan afforded four new limonoids with a C-19/C-29 bridged acetal structure, together with two known limonoids, 12α-hydroxyamoorastatone and its 12-acetate. The new compounds were elucidated as 1-O-acetyltrichilin H and 29-O-substituted amoorastatone derivatives, named neoazedarachins A, B and D, by spectroscopic and chemical means. Their antifeedant activity was also studied.
As a model experiment for the synthesis of optically active α,β-dibenzyl-α-hydroxy-γ-butyrolactone (1), (2S,3S)-2-benzyl-2-hydroxy-3-(3,4-methylenedioxybenzyl)-γ-buty-rolactone (3) was stereoselectively synthesized from L-(+)-arabinose via the carissanol-type of lignan, (2R/ S,3S,4S)-3-benzyl-2,3-dihydroxy-4-(3,4-methylenedio-xybenzyl)tetrahydrofuran (4).
Brassinosteroids in Japanese cedar (Cryptomeria japonica) were analyzed by GC-MS after being purified by reversed-phase HPLC. The occurrence of four stereoisomers of 23-dehydrobrassinolide, three stereoisomers of 28-homobrassinolide, and an isomer of homodolicholide was revealed. The stereochemical structures of these brassinosteroids remain undetermined. Among these, the 23-dehydrobrassinolide stereoisomers are the first brassinosteroids identified with an oxo group located at the C23 position. In addition, known brassinosteroids 3-dehydroteasterone, typhasterol and dolicholide were identified.
A convenient synthesis of the sialyl Lewis X (sLex) tetrasaccharide, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc (8), as a carbohydrate ligand for selectins is described. The key step is the reaction between NeuAcα2-3GalSMe (5) as a glycosyl donor and the suitably protected Fucα1-3GlcNAc derivative (4) as the glycosyl acceptor by using dimethyl(methylthio)sulfonium triflate (DMTST) as the promoter.
The structural diversity of the core lipids of extreme halophiles Haloarcula japonica and Halobacterium halobium was investigated. The most significant difference is that Ha. japonica contains sn-2,3-di-O-phytanylglycerol exclusively as the core lipid, whereas Hb. halobium contains both sn-2,3-di-O-phytanylglycerol and sn-2-O-sesterterpanyl (3,7,11,15,19-pentamethyleicosanyl)-3-O-phytanylglycerol.
Important intermediate 2 of the segment (3) possessing four sequential asymmetric centers (C9-C12) of aplysiatoxins was synthesized via dihydroxylation of 5 in only 7 steps from methyl (S)-3-hydroxy-2-methylpropionate. Another promising compound (10a) was also synthesized in only 6 steps via asymmetric dihydroxylation of chiral homoallylic alcohol 4 as the key step.
An immunological assay, based on the digoxigenin/anti-digoxigenin system, was developed to detect and quantify carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin (BSA) was oxidized by a hydroxyl radical-generating system consisting of ascorbate/Fe(III)/O2. The resulting albumin-derived carbonyls were labelled with digoxigenin-hydrazide and detected by dot blotting with an anti-digoxigenin antibody conjugated to alkaline phosphatase. Quantification was carried out by a densitometric analysi. This system allows the detection of a pmole-amount of carbonyl groups on blots. The assay covers a range of sensitivity from 1.26 to 126 pmoles. Another feature of this method is its application to a complex protein mixture (homogenate) to analyze the oxidative status of individual proteins, as are shown for intestinal brush border membrane homogenate of a rat.
By incubation with precursor amino acids, L-glutamate, L-cysteine, and glycine, or with L-cysteine, the unicellular cyanobacterium Synechocystis strain PCC 6803 increased intracellular glutathione levels, under illumination with white light. L-Cystine could not serve as the precursor. Two transport systems for L-cysteine were evident in the strain: a high (Ks; L-cysteine concentration giving one-half of the maximum uptake, 21~35 μM) and a low (Ks, 1.0~1.8 mM) affinity transport system. The latter, responsible for glutathione accumulation, was an energy-requiring process. L-Cysteine uptake by the two transport systems were Na+-independent, and inhibited by the presence of neutral amino acids and less inhibited by basic or acidic amino acids. These results suggested that the neutral amino acid symport(s) is involved in L-cysteine uptake for the GSH accumulation in this strain.
Catalase catalyzed the peroxynitrite-mediated nitration of 4-hydroxyphenylacetic acid. The curve for the pH dependence of nitration was similar to that for the reaction between peroxynitrite and phenol. Cyanide, azide, and 3-amino-1,2,4-triazole inhibited the nitration in a dose-dependent way. When catalase was mixed with peroxynitrite, Compound I was detected as an intermediate. Because azide was an electron donor for the peroxidatic action of catalase, and because 3-amino-1,2,4-triazole inhibited catalase activity by binding with Compound I, peroxynitrite-mediated phenolic nitration was probably accompanied by Compound I formation. Both catalase and superoxide dismutase protected Escherichia coli from peroxynitrite toxicity.
A very-high-density lipoprotein (VHDL) with a density of 1.27-1.29 g/ml was the most abundant lipoprotein in the hemolymph of the sand crayfish Ibacus ciliatus. The VHDL isolated by a density gradient ultracentrifugation consisted of 94% protein and 6% lipid reflecting its high density, and phospholipid was a predominant lipid component. The VHDL had an apolipoprotein of molecular mass 195 kDa and its N-terminal amino acid sequence was identified as follows: LQPGLEYQYRYNGRVAA. This sequence was similar to those of clotting proteins from the spiny lobster Panulirus interruptus and the freshwater crayfish Pacifastacus leniusculus. Transglutaminase and Ca2+ also induced the VHDL to clot. Considering large amounts of VHDL in the hemolymph of sand crayfish, the VHDL not only functions as lipid carrier but plays an important role in the defense process of crustacea.
A novel type of cysteine synthase (CSase, EC 220.127.116.11) isozyme, designated as CSase 1′, was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of Mr 34,000. The apparent Km for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1′ was maintained when it was treated at 60°C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, Km values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1′ were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.
Three cDNA clones encoding ADP-glucose pyrophosphorylases were isolated from a full red fruit cDNA library of watermelon (Citrullus vulgaris S.). Sequence analyses indicated that one clone, wms1, corresponds to the small subunit, and two clones, wml1 and wml2 (a partial gene), are the large subunits of AGPase. The presumed AGPase proteins encoded by wms1, wml1, and wml2 have 526, 526, and 481 amino acids, respectively. The protein sequences have the conserved amino acids important for the substrate or regulator binding site, with some variation. Developmental changes in the amounts of wms1, wml1, and wml2 transcripts in fruits were measured by northern blot analysis. Their expression levels decreased from the small green to medium green stages, then increased in accordance with fruit ripening, which was different from those of tomato and oriental melon.
A non-radioisotopic assay method for ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is described. 3-Phosphoglycerate produced by the RuBisCO reaction was measured using an anion-exchange chromatography. The reaction course of spinach RuBisCO measured by this method was completely the same as that measured with 14CO2.
Newly synthesized (1→3)-β-D-glucans with reducing glucose side chains (6-O-glucopyranosylated curdlan and 3-O-glucopyranosylated curdlan, with glucose linked directly (except for anomeric carbon) had antitumor activity against mice sarcoma 180 in mice. The two glucans potentiated the reticuloendotheliai system and activated macrophages (increased their glucose consumption). The activity inducing tumor regressing factor of the glucan derivatives was stronger than a linear (1→3)-β-D-glucan (curdlan).
An involution-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as full-length cDNA encoding the 40S subunit of ribosomal protein S14 (rps14). Comparison of the deduced amino acid sequence to sequences of rps14 from humans, hamsters, and rats showed a conservation.
Activity that inhibited protease of human immunodeficiency virus type 1 was found in boiling water extracts of an edible mushroom, Fuscoporia obliqua. The active component was identified as a water-soluble lignin derivative of high molecular weight. Other polyphenols of low molecular weight and monomeric components of lignin did not inhibit the protease.
Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.
A transgalactosylation reaction from p-nitrophenyl-α-D-galactopyranoside to 1-deoxynojirimycin was done using α-galactosidase [EC 18.104.22.168] from green coffee beans. The enzyme formed 6-O-α-D-galactopyranosyl-1-deoxynojirimycin as the major product.
To understand the structure-function relationship of an anionic lipopolysaccharide emulsan, the effect of hydrodynamic volume on the emulsifying activity was investigated. As a result, it was found that the hydrodynamic volume of emulsan was an important factor in its emulsifying activity. The hydrodynamic volume was decreased by the addition of a positively charged polypeptide, and the emulsifying activity was decreased, but negatively charged or uncharged polypeptide had little effect. These results suggest that the conformation of the backbone of emulsan helps to govern its emulsifying activity.
While examining the taste of various proteins, we found that hen egg-white lysozyme, a bacteriolytic enzyme, had sweetness. Lysozymes from other sources such as turkey and soft-shelled turtle also showed sweetness with different tastes, heavy or light. In contrast, human lysozyme was tasteless. The amino acid sequences of the various lysozymes were similar to that of hen lysozyme, but hen lysozyme did not show significant homology to sweet proteins.
HPLC analysis and yield of oleanene-glucuronide (OG) was done on some commercially available edible beans: seeds of Glycine max, Glycine max cv. Kuromame, Phaseolus vulgaris cv. Torosuku, Phaseolus vulgaris cv. Toramame, Phaseolus vulgaris cv. Taishokintoki, Phaseolus coccineus cv. Ooshirobana, Phaseolus coccineus cv. Murasakihanamame, Vigna unguiculata cv. Chuguro, Vigna angularis cv. Dainagon, Arachis hypogaea, Pisum sativum, and Vicia faba. All the beans listed above have OG, though in varying the amounts. Furthermore the HPLC profiles of beans belonging to the same genus were very similar except for that of the Vigna genus. There was no great difference of the HPLC profiles with respect to the cultivated varieties. The structures of the major OGs in each type of beans were identified as soyasaponins I (1) and V (2), and phaseoside I (3) (Phaseolus vulgaris and P. coccineus); 1 and soyasaponin II (4) (Glycine max); 1 and 2 (Vigna unguiculata); 1 (Pisum sativum, Arachis hypogaea and Vicia faba). In contrast, those in Dainagon (Vigna angularis cv. Dainagon) were identified as azukisaponins II (5) and VI (6).
Subset analysis of splenic lymphocytes using flow cytometry showed that the percentages of Thy1.2-(pan T-cells), L3T4-(CD4, helper T-cells), and Lyt2-(CD8, cytotoxic T-cells) positive cell populations were significantly increased in mice orally administered a hot water-soluble fraction from Agaricus blazei as compared with mice treated only with saline. 13C-NMR data indicates that the main component in the active polysaccharide is the complex of α-1,6- and α-1,4-glucan, which had already been shown to have anti-tumor activity against Sarcoma 180. It seems that the polysaccharide from Agaricus blazei may be an effective prophylactic, protecting humans against cancer by stimulating lymphocytes such as cytotoxic T-cells.
The effects of natural and non-natural fatty acids on enhancing the absorption of a hydrophilic marker through a human epithelial cell (Caco-2) monolayer were measured to elucidate the properties of the fatty acids. Fatty acids from C9 to C14 enhanced the absorption depending on the concentration and the carbon chain length. Those fatty acids with longer chains gave a higher permeability coefficient at low concentrations and a lower toxicity than those with shorter chains. The surface energy lowering coefficient (SELC), an intrinsic physico-chemical property, and the critical micellar concentration (CMC) were good criteria for identifying the threshold concentrations of a fatty acid to significantly enhance absorption.
The molecular inclusion powder of d-limonene in β-cyclodextrin was prepared by using a twin-screw kneader at a low water content. The influence of water and alcohol content on the formation of the inclusion complex was studied in comparison with the inclusion complex obtained by the micro-aqueous method. The inclusion fraction of d-limonene in the complex powder in β-cyclodextrin was much higher than that made by the micro-aqueous method. The marked differences in the inclusion fraction were observed particularly at the molar water ratio to β-cyclodextrin of less than 10. The inclusion fraction increased sharply with the increase in the kneading torque. This means that the energy of kneading increased the inclusion of d-limonene. A mathematical model for estimating the inclusion fraction was derived by combining the effects of water and the kneading energy on the formation of the inclusion complex.
To study the mechanism of the fall of glutathione peroxidase (GSH-Px) activity in erythrocyte after cerebral strokes in stroke-prone spontaneously hypertensive rats (SHRSP), erythrocytes were fractionated into low density erythrocytes (LD-E) and high density erythrocytes (HD-E) by a density gradient centrifugal method using Percoll solution, and fluctuation of the distribution ratio and changes of GSH-Px activity in fractionated erythrocytes were investigated. The distribution ratio of LD-E and HD-E in erythrocytes of SHRSP was about 4:1 at 5 weeks of age (n=6), and the distribution to HD-E increased along with aging. While the distribution ratio was changed, however, there was no change in the GSH-Px activity in both LD-E and HD-E of erythrocytes. In senile, 30-week-old SHRSP (n=4) with advanced hypertension, the GSH-Px activity in the HD-E was lower, in proportion to the increase of the distribution rate against HD-E. On the other hand, in SHRSP (n=5) having cerebral stroke, the distribution ratio of LD-E and HD-E was about 1:4. The GSH-Px activity was 31.4±2.9 units/1010 erythrocytes in LD-E, which was hardly different from the value of SHRSP without stroke (35.7±3.3 units/1010 erythrocytes). In HD-E, however, the activity was 18.2±2.2 units/1010 erythrocytes, being lower than the activity of SHRSP without stroke. At the moment when the GSH-Px activity had dropped to 17 units/mg hemoglobin, and the control diet was changed to one based on fish or a hydralazine treatment given, the activity recovered, and an increase in body weight and the distribution rate of the LD-E over HD-E was increased. It is clear from these experiments that the fall of erythrocyte GSH-Px activity observed after cerebral stroke is due to a decrease of LD-E and increase of HD-E, which has lowered activity. However, nothing definite is known on the relationship between the fall of GSH-Px activity in erythrocytes and disorder in cerebral tissue. It appears that the fall of the GSH-Px activity causes at least functional and structural changes in erythrocytes, which interfere with the delivery of oxygen to peripheral tissues, triggering oxidation stress in cerebral tissues.
Orotic acid is known to cause fatty liver, but it is unclear whether this is caused partly by stimulation of the enzymes for triacylglycerol (TG) synthesis. To understand the change of hepatic TG metabolism in fatty liver induced by orotic acid, we determined the liver tissue TG level and phosphatidate phosphohydrolase (PAP) activity over time in rats fed on a diet containing orotic acid (OA). A dietary lipid content of 10% was achieved by using n-6 fatty acid-rich corn oil in experiment 1, and n-6 fatty acid-rich safflower oil (SO) and n-3 fatty acid-rich fish oil (FO) with the same polyunsaturated fatty acid/monounsaturated fatty acid/saturated fatty acid (P/M/S) ratio in experiment 2. In experiment 1, an increase in the hepatic TG level due to OA intake was observed from day 5 onwards, the level rising approximately 6-fold by day 10. The activity of hepatic microsomal PAP, the rate-limiting enzyme in TG synthesis, increased markedly from day 5 onwards, concurrent with the liver diacylglycerol concentration. A strong correlation (r=0.974) was observed between the hepatic TG level and microsome-bound PAP activity. In experiment 2, we investigated the effects of dietary fatty acid on OA-induced fatty liver. Compared with the n-6 fatty acid-rich vegetable oil diet, the relative increase in hepatic TG was smaller with the n-3 fatty acid-rich FO diet, and hepatic PAP activity fell markedly to the level for an OA-free diet. In addition, the hepatic TG accumulation and serum TG concentration were lower in the FO group than in the SO group. Nevertheless, because the hepatic TG level was low, it seems that the inhibition of liver PAP activity by FO possibly had a strong influence on the accumulation of TG in the liver. In conclusion, enhanced TG synthesis mediated by changes in liver PAP activity was involved in the hepatic TG accumulation induced by OA administration, this change being markedly suppressed by dietary n-3 fatty acids.
An aqueous methanol extract from green tea showed potent acetyl-CoA carboxylase inhibitory activity. An active compound was isolated from the extract and identified as (-)-epigallocatechin gallate by instrumental analyses. The IC50 value of (-)-epigallocatechin gallate was 3.1×10-4 M. Among tea catechins and related compounds, nearly equal activity was found in (-)-epigallocatechin gallate and (-)-epicatechin gallate, whereas (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, gallic acid and methyl gallate each had no inhibitory activity. These results indicate that the 3-O-gallate group of the catechin structure was necessary for this activity. (-)-Epigallocatechin gallate inhibited triglyceride accumulation in 3T3-L1 cells at a concentration of 1.0×10-7 M or higher.
We determined whether the synthesis and degradation of N-acetylglutamate would regulate urea synthesis when the thyroid status was manipulated. Experiments were done on three groups of rats, each being given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without a triiodothyronine (T3) treatment, treated with PTU+T3, or receiving neither PTU nor T3 (control). The plasma concentration and urinary excretion of urea, the liver concentration of N-acetylglutamate, and the liver N-acetylglutamate synthesis in rats given PTU alone were each significantly higher than in the control rats. Compared with the control rats, the liver N-acetylglutamate degradation was significantly lower in those rats given PTU without the T3 treatment. Treatment of the PTU-treated rats with T3 reversed the effects of PTU to the values of the control rats. N-Acetylglutamate synthesis in the liver was closely correlated with the excretion of urea, and inverse correlation between the liver N-acetylglutamate degradation and urea excretion was found. These results suggest that the greater synthesis and lower degradation of N-acetylglutamate in the hypothyroid (PTU alone) rats would be likely to increase the hepatic concentration of this compound and stimulate urea synthesis.
Gastric acid secretion is suppressed, resulting in a significant rise in gastric pH, when conscious rats are exposed to hypoxia (Yamaji et al., 1996). When adrenal medullectomized rats were exposed to moderate (10.5% O2) hypoxia for 3 h, gastric acid secretion was restored to nearly the level in normoxia by the adrenal medullectomy. In severe (7.6% O2) hypoxia, the operation also caused an increase in the level of gastric acid output, although the extent was lower than that under 10.5% O2 hypoxic conditions. Gastric pH was normalized by the operation even with 7.6% O2 hypoxia. Similar results were obtained when reserpine, which causes an adrenergic discharge, was administered. When an α2-adrenoceptor blocking agent, yohimbine, was administered, the inhibitory effect of 10.5% and 7.6% O2 hypoxia on gastric acid secretion was almost completely removed. However, neither prazosin (an α1-adrenoceptor blocker) nor propranolol (a β-adrenoceptor blocker) showed any significant effects on gastric acid output in hypoxia. These results indicate that acute hypoxia stimulated the adrenergic response from the adrenal medulla, and that gastric acid secretion was consequently suppressed through α2-adrenoceptor.
To examine the antioxidative compounds of nonprotein amino acids in a red alga, Porphyra yezoensis, an ethanol extract of the cultured thalli was fractionated with Dowex columns. The basic fraction V showed strong antioxidative capacities with ferric thiocyanate and TBARS measurements. In this basic fraction, histidine, 3-methylhistidine, carnosine, and anserine were detected beside ornithine, lysine, and arginine by amino acid analysis. The occurrence of carnosine and anserine suggests that the histidine-related compounds also contribute to the antioxidative reactions in P. yezoensis.
Taurine, 2-amino ethanesulfonic acid, is the major free intracellular amino acid present in many tissues and plays an important role in lipid metabolism such as that of bile acid conjugation for fat absorption. The effect of taurine on the serum cholesterol level in normal rats was investigated. Taurine enhanced the serum HDL-cholesterol concentration in a dose-dependent manner without any change in total cholesterol.
The effect of food restriction on the conversion ratio of tryptophan to niacin was investigated, because it is known that the conversion ratio is influenced by nutritional factors. A 20% casein diet was fed to rats ad libitum (control), 1/2 the food of the control. 1/4 the food of the control, or starved for 9 days, and urine samples were collected to measure the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, and nicotinamide. The conversion ratio in the 1/2, 1/4, or starving group increased at day 1 of the experiment, but returned to the original value from day 2. Only in the starving group did the conversion ratio extremely increase from day 6 to day 9, being about 5-times higher than that of the original value on day 9. The possible mechanism by which the conversion ratio increased during food restriction is discussed.
Egg white forms a soft opaque gel at around 65°C. An analysis of the turbidity appearance in the presence or absence of iron and polyacrylamide gel electrophoresis of the precipitated proteins after thermal treatment revealed ovotransferrin to be the major component involved in thermal gelation at around 65°C. The storage modulus of the thermally induced gel of purified ovotransferrin reinforced this conclusion.
Dietary supplementation with powder of a green tea extract suppressed the enhancement of plasma alanine aminotransferase and aspartate aminotransferase activities induced by D-galactosamine, but not by carbon tetrachloride, in a dose-dependent manner in rats. The minimum dose to cause a significant effect was 1 to 2%. Drinking green tea also suppressed plasma enzyme activities. These results indicate that green tea had a liver injury-preventive effect.
Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.
Hydroxy isothiocyanates (ITCs), including some new derivatives of naturally occurring compounds, were synthesized and their minimum inhibitory, minimum fungicidal, and minimum bactericidal concentrations for Aspergillus niger, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli were estimated. These compounds were strongly antimicrobial; for example, 2-(4-hydroxyphenyl)ethyl ITC inhibited growth of all strains examined at concentrations of 7.8 to 15.6 μg/ml. The ATP concentration in E. coli was markedly reduced when cells were treated with 2-(4-hydroxyphenyl)ethyl ITC. Inhibition of the growth of E. coli by 2-(4-hydroxyphenyl)ethyl ITC was decreased in the presence of cysteine. Streptolysin S production in washed cells of Streptococcus equisimilis was extremely sensitive to this ITC derivative and this inhibition also was counteracted by cysteine. The results showed that the ITC compounds had antimicrobial effects by blocking sulfhydryl groups.
A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking. This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242. The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19. The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides. Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band. The results suggested that the sugars accounted for the differences in the molecular masses. The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe- Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala- Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr. Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme. Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E. coli after translation. The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.
An acidic-phospholipid deficiency caused by the pgsA3 allele that encodes a defective phosphatidylglycerophosphate synthase in Escherichia coli is lethal. The only known mutations that suppress this lethality fully have been related to the major outer-membrane lipoprotein. We isolated a Bacillus subtilis chromosomal locus that suppresses the lethality when harbored in a low copy-number plasmid, without restoring the synthase activity or phospholipid composition to normal. The locus was first recognized to suppress the conditional lethality of E. coli YA5512 (pgsA3) that harbored an unidentified mutation(s), allowing its growth in LB medium but not in media of lower osmolarities. The locus was then found to suppress the lethality of pgsA3 in wild-type E. coli W3110. This locus, named ypoP in the database, had 37% nucleotide identity with the E. coli mprA gene, but the amplification of mprA had no suppressive effect. Plasmid pPOP1 containing ypoP completely prevented the decrease in the amount of a porin protein, OmpF, in the outer membrane and also cell mucoidy caused by pgsA3. The mechanisms underlying these unusual effects are discussed in relation to a putative stress signal(s) generated by the acidic-phospholipid deficiency.
We purified scytalone dehydratase from the rice blast fungus, Pyricularia oryzae, and cloned its cDNA on the basis of its amino acid sequence. The deduced amino acid sequence was 62% identical to the scytalone dehydratase from Colletotrichum lagenarium. The expression of this gene was induced transcriptionally in the stationary phase when melanin synthesis occurs. We constructed a heterologous expression system for the enzyme in Escherichia coli, did deletion analysis with this system, and found that a C-terminal region is essential for the enzyme function.
The effects of dibromochloropropane (DBCP), a pesticide, on the male rat reproductive system were examined at morphological and hormonal expression levels. Changes of Leydig cells and seminiferous tubules were examined in rats treated with one subcutaneous (s.c.) injection of DBCP at doses of 10, 50, 75 and 100 mg/kg of body weight. The Leydig cells degenerated and decreased in number in those rats with an s.c. injection of DBCP at a dose of 50 mg/kg of body weight. However, these morphological changes were not significant in those rats that were treated with four separate s.c. injections of DBCP at 10 mg/kg of body weight. The expression of luteinizing hormone receptor mRNA, which is specifically expressed in Leydig cells, was decreased significantly in the DBCP-treated rats as compared with the normal-control rats. The expression of follicle-stimulating hormone receptor mRNA was decreased to a mild degree in DBCP-treated rats. At a dose of 75 mg/kg of body weight, seminiferous tubules as well as Leydig cells were severely damaged morphologically and at hormonal receptor expression levels. Multinucleated giant cells appeared at a dose of 75 mg/kg of body weight. All the rats died at a dose of 100 mg/kg of body weight. Our data indicate that DBCP had a cytotoxic effect on the male rat reproductive system in a dose-dependent manner.
In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 22.214.171.124) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.