Three cyclohexenone derivatives, (4S,5S,6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (1), (4R,5R)-4,5-dihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (2), and (4R,5S,6R)-4,5,6-trihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (3), were isolated from unpolished rice fermented with an xylariaceous endophytic fungus (strain YUA-026). The structures of three compounds were established on the basis of spectroscopic analyses and chemical conversion. The minimum inhibitory concentrations of 1 and 3 were 100 μg/ml and 400 μg/ml against Staphylococcus aureus, 100 μg/ml and 200 μg/ml against Pseudomonas aeruginosa, and 200 μg/ml and >400 μg/ml against Candida albicans, respectively. In addition, 1 and 3 exhibited phytotoxic activity against lettuce.
The biosynthesis of pamamycin-607 (PM-607), a sixteen-membered macrodiolide compound, was studied with 13C- and 15N-labeled precursor units in Streptomyces alboniger. Feeding experiments with 13C-labeled acetate or propionate indicate that the carbon skeleton of PM-607 was derived from six acetate, four propionate and three succinate units. MS analyses of 15N-labeled PM-607 suggest that the nitrogen atom in PM-607 was derived from the α-amino group of an amino acid.
Both enantiomers and the racemate of α-pinene were transformed by Picea abies cells immobilised on alginate. The main products were cis- and trans-verbenol, the later being further transformed to verbenone. The enantiomeric purity of each product more or less corresponded to that of the substrate. Transformation by free cells was faster than that by the immobilised cells. The ratio of products differed to some extent between the transformation by free and immobilised cells.
A new ergothioneine derivative named β-hydroxyergothioneine was isolated from the mushroom Lyophyllum connatum. Ergothioneine,N-hydroxy-N′,N′-dimethylurea, and connatin (N-hydroxy-N′,N′-dimethylcitrulline) were also isolated. All the compounds displayed the ability to scavenge free radicals, based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Structural determination, including the absolute stereochemistry of β-hydroxyergothioneine, was achieved by spectroscopic analysis and X-ray crystallography. The radical scavenging activity of β-hydroxyergothioneine was almost the same as that of ergothioneine. β-Hydroxyergothioneine showed the greatest protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes.
The first enantioselective total synthesis of enokipodins A, B, C and D, highly oxidized α-cuparenone-type sesquiterpenoids possessing antimicrobial activity, was accomplished in 8–28% overall yields from methyl (2,5-dimethoxy-4-methylphenyl)acetate by applying Meyers’ diastereoselective alkylation protocol for the construction of their C7-quaternary asymmetric center. The present synthesis confirmed the absolute configuration of the enokipodins, and also constitutes a formal enantioselective synthesis of (S)-1,4-cuparenediol and (S)-cuparene-1,4-quinone.
Studies using genetics and vitamin A deficiency (VAD) have shown that vitamin A and retinoids play essential roles in spermatogenesis at the pre-meiotic stage. To understand the mechanisms of control in spermatogenesis by retinoic acid, we investigated whether retinoic acids regulate the expression of downstream transcription factors that are essential for spermatogenesis. In this study, we found that administration of all-trans retinoic acid (ATRA) or retinol to VAD rats down-regulates the testicular mRNA levels of the cAMP responsive element modulator (CREM), an essential transcription factor for spermatogenesis. Conversely, depletion of retinoids from the diet leds to an up-regulation of CREM expression in adult testes. In addition, RT-PCR analysis indicated that ATRA specifically represses the expression of the activator spliced variant of CREM (CREMτ). These results suggest that retinoids function as a negative regulator of CREM expression in testes.
Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.
Gibberellins A1⁄3 (GA1⁄3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1⁄3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1⁄3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible α-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.
A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS–PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.
Recently, we found that vaticanol C (a resveratrol tetramer), which was isolated from stem bark of Dipterocarpaceae, exhibited growth suppression and induction of apoptosis via the loss of mitochondrial membrane potential and consequent caspases activation. The detailed mechanisms are not clearly understood. We decided to attempt to gain further insight into the mechanisms underlying vaticanol C-induced apoptosis in HL-60 cells. Treatment of HL-60 cells with vaticanol C was found to cause a marked decrease in the level of phosphorylated extracellular signal-regulated kinase (ERK) concurrent with inhibited phosphorylation of its upstream kinase mitogen-activates protein kinase kinase (MEK). Moreover, exposure to vaticanol C led to a significant reduction in the level of phosphorylated Akt. Thus, vaticanol C induced inhibition of both ERK and Akt phosphorylation, resulting in reduced phosphorylation of Bad. These results suggest that vaticanol C might induce apoptosis via a mechanism involving activation of Bad through disruption of pro-survival signaling pathways.
The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 °C, lower than that of E. coli APase by 30 °C. The specific activity of SIB1 APase at 50 °C was 3.1 fold higher than that of E. coli APase at 80 °C. SIB1 APase lost activity with a half-life of 3.9 min at 70 °C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 °C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.
In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9) is interesting because the most probable clock component TIMING OF CAB EXPRESSION 1 (TOC1) belongs to this family. Several lines of evidence have already been accumulated to support the view that not only APRR1/TOC1 but also other APRR family members are crucial for a better understanding of the molecular link between circadian rhythm and light-signal transduction. Among the APRR1/TOC1 family members, the circadian-controlled APRR9 gene is unique in that its expression is rapidly induced by light at the level of transcription. In this study we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87 cells). Taking the results of these approaches together, we provide several lines of evidence that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: an “L element” responsible for the light-induced expression, followed by an “R element” necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.
Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.
A cDNA clone of a novel cytochrome P450, CYP76A4, was isolated from Petunia hybrida. The cDNA clone contained an open reading frame (ORF) encoding a predicted 510 amino acid polypeptide. The CYP76A4 cDNA was expressed in yeast Saccharomyces cerevisiae AH22. Recombinant yeast microsomes containing the CYP76A4 hemoprotein were found to catalyze (ω-1)-hydroxylation of lauric acid.
A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.
RNA interference is a powerful technique for suppressing gene functions in many eukaryotes including plants. Here we show that introduction of double-stranded RNA into Arabidopsis protoplasts leads to marked silencing of endogenous genes, as observed previously for transgenes [Biosci. Biotechnol. Biochem., 67, 2674–2677 (2003)]. This simple system should be useful for functional analysis of genes involved in fundamental cellular processes.
The complete nucleotide sequence for pTB6 (3,624 bp) from Bifidobacterium longum was determined. This plasmid is 95% homologous in nucleotide (nuc) sequence, and also 92% in RepB aa sequence, to rolling circle replication (RCR) plasmids pKJ36 and pB44, suggesting that pTB6 replicates by the rolling circle mechanism. The putative MembB, MobA, and protein encoding from orf (Orf) I detected were nonessential for plasmid replication. We constructed an immobile shuttle vector from pTB6 and pUC18, which transformed B. longum with a high efficiency of 2.5×106 transformants/μg DNA.
In general, the clock (or oscillator) is central to circadian rhythms in many organisms. In the model higher plant Arabidopsis thaliana, the best candidates forclock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL), which are homologous Myb-related transcription factors. It is also believed that TOC1 (TIMING OF CAB EXPRESSION 1) is another component of the central oscillator. In this connection, we have been characterizing a small family of proteins, designated ARABIDOPSIS PSEUDO-RESPONSE REGULATOR (PRR1, PRR3, PRR5, PRR7, and PRR9), based on the fact that one of the members (PRR1) is identical to TOC1. Nevertheless, it is not yet certain whether other PRR family members are also implicated in clock function per se. To address this issue, in this study we examined a functional interaction between the CCA1 clock component and one of the PRR family members, PRR5, by employing transgenic lines overexpressing both the CCA1 and PRR5 genes. Evidence will be provided that PRR5 plays an antagonistic role(s) to the putative CCA1 clock component.
Sufu is a popular fermented tofu product in China. The low quality of sufu produced in the hot summer is a big problem in sufu manufacture, so we prepared sufu at two different temperatures, 26 °C as normal and 32 °C as high temperature, and the effects of temperature on isoflavones and β-glucosidase activity were investigated. Fermentation temperature did not cause significant differences in the recovery of isoflavones, but resulted in a different redistribution of isoflavone isomers in sufu. Sufu fermented at 26 °C was richer in isoflavone aglycones than at 32 °C; the enrichment of isoflavone aglycones might have the advantage of enhancing the physiological function. No 6″-O-malonyl-glucosides were detected in sufu fermented at 26 °C, whereas some 6″-O-malonyl-glucosides were found at 32 °C. A fermentation temperature of 26 °C benefited the β-glucosidase production by fungi, which contributed to valid conversion from β-glucosides to aglycones. It was also found that β-glucosidase converted β-glucosides more effectively than 6″-O-malonyl-glucosides and 6″-O-acetyl-glucosides into aglycones.
It is known that niacin itself is not necessary in rats when tryptophan is given in adequate amounts, because rats can biosynthesize niacin from tryptophan. In our experiment, young rats were fed on a 20%, 40%, 60%, or 70% casein diet with or without niacin. The rats fed on the 20%, 40%, and 60% casein diets did not require niacin for growth, but the rats fed on the 70% casein diet needed it. This phenomenon was attributed to the supposition that liver aminocarboxymuconate–semialdehyde decarboxylase activities increased according with the dietary casein levels. The conversion ratio of tryptophan–niacin in rats fed on the 70% casein diet became extremely low, and then the rats needed niacin.
Two anthocyanins, cyanidin-3-α-O-rhamnoside (C3R) and pelargonidin-3-α-O-rhamnoside (P3R), and quercitrin (quercetin-3-α-O-rhamnoside), were isolated from acerola (Malpighia emarginata DC.) fruit. These polyphenols were evaluated based on the functional properties associated with diabetes mellitus or its complications, that is, on the radical scavenging activity and the inhibitory effect on both α-glucosidase and advanced glycation end product (AGE) formation. C3R and quercitrin revealed strong radical scavenging activity. While the inhibitory profiles of isolated polyphenols except quercitrin towards α-glucosidase activity were low, all polyphenols strongly inhibited AGE formation.
Previous studies have suggested that docosahexaenoic acid (DHA), contained in fish oil, prevents brain disease. In the current study, the effect of fish oil feeding on gene expression in the brain was investigated by suppression subtractive hybridization. We found that pyruvate dehydrogenase E1 alpha (PDHE1α) mRNA expression is down-regulated by fish oil feeding. We examined whether the expression of PDHE1α mRNA is altered by DHA treatment in differentiated PC12 cells. PDHE1α mRNA was reduced by supplementation of DHA with a significant decrease in cellular ATP level. These results indicate that fish oil feeding might modulate energy metabolism in the brain.
Overexpression of matrix metalloproteinases (MMPs) is associated with cancer metastasis. We assessed mRNA expression of MMPs in six human colorectal cancer cell lines and found a considerable level of MMP-7 expression in HT-29 cells. Next, we searched for natural and synthetic compounds that cause a reduction in the production of proMMP-7 protein, and found that nobiletin (NOB), quercetin, valeryl salicylate, and sulindac sulfone demonstrated marked inhibition. Importantly, NOB attenuated proMMP-7 protein and its mRNA expression both concentration- and time-dependently via a reduction of activator protein-1 (AP-1) DNA binding activity, suggesting it as a promising agent for suppression of cancer cell invasion and metastasis through MMP-7 gene repression.
Three types of straight doughs different in combination of yeast and shortenings (RLS20, FTS20, and FTS80) were prepared, and the structure of the frozen doughs was examined under a microscope after staining protein or lipid droplets. Even after 2 months of frozen storage, distinct changes were not found in the gluten network of FTS80, although significant damages in the dough structures of FTS20 and RLS20 appeared after only one month of frozen storage. These results suggest that the gluten networks loosen and decrease in the water retention ability, and it may be concluded that the lipid is removed from the gluten protein due to the decrease in water in the continuous protein phase. The resulting product from the damage to the gluten matrix gave rise to fusion of lipid droplets and an increase in their size. Because of the difference in fatty acid composition, the lipids of shortening S80 are presumed to interact more strongly with gluten proteins and to keep the gluten matrix from damage in comparison with the lipids of shortening S20.
This report describes a green chemical method for controlling soil-borne plant diseases without disinfection using an equimolar copolymer of N-benzyl-4-vinylpyridinium chloride with styrene (PBVP-co-ST) that captures microbial cells alive on the surface and is highly biodegradable. Tomato bacterial wilt caused by Ralstonia solanacearum was controlled by the addition of sawdust coated with PBVP-co-ST prior to transplantation. This effected 87% reduction in appearance and 89% reduction in the index of symptom under appropriate conditions. The coated sawdust did not exhibit bactericidal activity. The half-life of PBVP-co-ST was 5.6 d when treated with activated sludge in soil. The disease control was explained in terms of reduction of infectious contact between the roots of tomato and the cells of R. solanacearum due to coagulation-like interaction between microbial cells and the coated sawdust, in addition to capture of microbial cells by the coated sawdust.
We purified and characterized a cold-active polygalacturonase (PG) from the extracellular fraction of Cystofilobasidium capitatum strain PPY-1. The purified PG from strain PPY-1 has a molecular mass of about 44 kDa, and exhibited high activity at 0 °C, although its optimum temperature was 45 °C. Although the Km value for polygalacturonate as a substrate at 45 °C was found to be 11.2 mg/ml, it decreased gradually with decreasing temperature, and it was 0.66 mg/ml at 0 °C. Moreover, its cleavage pattern was of the endo-type. These findings might indicate that PG from strain PPY-1 is a novel type of cold-active endo-PG that is able to degrade pectin compounds at low temperatures.