Calcineurin, a highly conserved Ca2+/CaM-dependent protein phosphatase, plays key regulatory roles in diverse biological processes from yeast to humans. Genetic and molecular analyses of the yeast model system have proved successful in dissecting complex regulatory pathways mediated by calcineurin. Saccharomyces cerevisiae calcineurin is not essential for growth under laboratory conditions, but becomes essential for survival under certain stress conditions, and is required for stress-induced expression of the genes for ion transporters and cell-wall synthesis. Yeast calcineurin, in collaboration with a Mpk1 MAP kinase cascade, is also important in G2 cell-cycle regulation due to its action in a checkpoint-like mechanism. Genetic and molecular analysis of the Ca2+-dependent cell-cycle regulation has revealed an elaborate mechanism for the calcineurin-dependent regulation of the G2/M transition, in which calcineurin multilaterally activates Swe1, a negative regulator of the Cdc28/Clb complex, at the transcriptional, posttranslational, and degradation levels.
The genome sequence of Aspergillus oryzae, a fungus used in the production of the traditional Japanese fermentation foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste), has revealed prominent features in its gene composition as compared to those of Saccharomyces cerevisiae and Neurospora crassa. The A. oryzae genome is extremely enriched with genes involved in biomass degradation, primary and secondary metabolism, transcriptional regulation, and cell signaling. Even compared to the related species A. nidulans and A. fumigatus, an abundance of metabolic genes is apparent, with acquisition of more than 6 Mb of sequence in the A. oryzae lineage, interspersed throughout the A. oryzae genome. Besides the various already established merits of A. oryzae for industrial uses, the genome sequence and the abundance of metabolic genes should significantly accelerate the biotechnological use of A. oryzae in industry.
The relationship between antibacterial activity of tetra-substituted tetrahydrofuran lignans (1–4) and their absolute configurations was tested. Only compound 4 among two virgatusins and two related compounds exhibited growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Listeria denitrificans. Compound 4 affected the growth of B. subtilis in a bactericidic manner, and its ability to dissipate the cytoplasmic membrane potential was investigated using the fluorescence probe 3,3′-dipropylthiadicarbocyanine iodide. These results suggested that compound 4 damages cells by causing the loss of the proton motive force and disruption of the cellular integrity of the membrane, leading to cell death. In addition, it was shown that the antibacterial activity of a lignan was closely related to its absolute configuration and functional groups.
We have previously isolated cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) from an actinomycete by a novel enzymatic conversion-guided method. Their tetradehydro derivatives, cyclo(ΔPro-ΔTyr) and cyclo(ΔPhe-ΔPro), were enzymatically prepared. Neither of them inhibited cell division, in contrast to other tetradehydro cyclic dipeptides prepared previously. This result suggests that an NH proton in a diketopiperazine ring and/or conformation of the compound are important for the activity.
Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.
Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.
UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to the respective UDP-sugars in the salvage pathway. Using the genomic database, we cloned a putative gene for UDP-sugar pyrophosphorylase from Arabidopsis. Although relatively stronger expression was detected in the vascular tissue of leaves and the pollen, AtUSP is expressed in most cell types of Arabidopsis, indicating a housekeeping function in nucleotide sugar metabolism. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates, resulting in the formation of various UDP-sugars such as UDP-glucose, -galactose, -glucuronic acid, -xylose and -L-arabinose. A loss-of-function mutation in the AtUSP gene caused by T-DNA insertion completely abolished male fertility. These results indicate that AtUSP functions as a UDP-sugar pyrophosphorylase in the salvage pathway, and that the generation of UDP-sugars from monosaccharide 1-phosphates catalyzed by AtUSP is essential for pollen development in Arabidopsis.
The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both α-factor and Ste4 Gβ overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.
A cDNA for NADH–cytochrome b5 reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5′- and 3′-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent Km values of 2 μM and 14 μM for NADH and cytochrome b5 respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b5 reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.
Hydrophobic bile acids but not hydrophilic bile acids induce apoptosis in HCT116 cells. We expressed sodium-dependent bile acid transporters in HCT116 cells, and the intracellular concentration of hydrophilic bile acids increased to that of the hydrophobic bile acids. But no sign of apoptosis was observed, which suggests a hydrophobic-bile acid-specific mechanism for the induction of apoptosis in HCT116 cells.
Cauxin is a carboxylesterase-like glycoprotein excreted as a major component of cat urine. Cauxin contains four putative N-glycosylation sites. We characterized the structure of an N-linked oligosaccharide of cauxin using nano liquid chromatography (LC)-electrospray ionization (ESI) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) and MS/MS, and high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column. The structure of the N-linked oligosaccharide of cauxin attached to 83Asn was a bisecting complex type, Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)(GlcNAcβ1-4)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.
To establish efficient induction of Cre mediated DNA recombination in primary cells, mouse embryonic fibroblast, keratinocyte, and primary preosteoblast, we tested various recombinant Cres by fusing of protein transduction domain in human immunodeficiency virus (HIV) transactivator of transcription (TAT-PTD) to the N- and/or C-terminus. HTC, modified Cre with PTD at the N-terminus, achieved the highest activity of DNA recombination for those primary cells.
In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.
The neuropeptide Orexin is involved in the regulation of the sleep-awake cycle and feeding behavior. We isolated a 22-kb genomic clone containing the 5′ flanking region of the mouse Orexin promoter. We determined that the transcription start site (+1) is located 96 nucleotides upstream of the initiation codon. The start site region contained consensus sequences corresponding to the transcription initiator and TATA box. Analysis of promoter activity using PC12 cells showed that regions between −13 and +112 and between −1,868 and −780 contained nerve growth factor (NGF)-responsive positive regulatory element and a negative regulatory element respectively.
We investigated the changes in autonomic nervous activity, body temperature, blood pressure (BP), and heart rate (HR) after intake of the non-pungent pepper CH-19 Sweet and of hot red pepper in humans to elucidate the mechanisms of diet-induced thermogenesis (DIT) due to CH-19 Sweet. We found that CH-19 Sweet activates the sympathetic nervous system (SNS) and enhances thermogenesis as effectively as hot red pepper, ant that the heat loss effect due to CH-19 Sweet is weaker than that due to hot red pepper. Furthermore, we found that intake of CH-19 Sweet does not affect systolic BP or HR, while hot red pepper transiently elevates them. These results indicate that DIT due to CH-19 Sweet can be induced via the activation of SNS as well as hot red pepper, but that the changes in BP, HR, and heat loss effect are different between these peppers.
We examined the effects of a nondigestible disaccharide difructose anhydride III (DFAIII) on calcium absorption and retention by means of a human balance study of single-blind crossover design. Twelve healthy male subjects ingested 250 mg of shell powder as calcium carbonate (corresponding to 100 mg of calcium) with or without 1.0 g DFAIII three times a day for 13 d. In the last 4 d as a balance period, all urine and feces were collected and evaluated for calcium excretion. The apparent calcium absorption (mg/d) and rate of absorption (%) were higher, and those of retention were much higher, in the DFAIII group than in the control group. Furthermore, serum osteocalcin increased after the experimental period in the DFAIII group but not in the control group. These results indicate that DFAIII ingestion enhances intestinal calcium absorption, which might be beneficial for bone metabolism.
The effect and mechanism of two types of rice protein, one from regular japonica rice Koshihikari and another from rice cultivar Shunyo, with low glutelin and high prolamin content, on cholesterol and triglyceride metabolism were compared by feeding casein and soy protein to male Wistar strain rats 7 and 20 weeks old ad libitum for 2 weeks. The results in adult rats clearly indicated that both rice proteins had cholesterol-lowering effects in the plasma and the liver, comparable to soy protein, and the effects were accompanied with TG-lowering effects in the liver. Similar effects were also observed in growing rats when the diets were supplemented with cholesterol. The mechanism of the cholesterol-lowering effects by these rice proteins cannot be explained solely by fecal steroid excretion, but the results indicate that not only regular rice protein but also Shunyo rice protein possesses improving effects on lipid metabolism, especially in the adult period.
High-molecular-weight polyphenols from oolong and black teas increased mitochondrial membrane potential, as measured by a method using ciliated protozoan Tetrahymena and rhodamine 123. These polyphenols, referred to as mitochondrial activation factors (MAFs), were purified from oolong and black teas by solvent extraction and Toyopearl column chromatography. The number-average molecular weights of the MAFs were 9,000 to 18,000, and the weight-average molecular weights were 15,000 to 25,000. The MAFs increased the mitochondrial membrane potential more than catechins did. Treatment of the MAFs with tannase indicated that they contained galloyl residues. When the MAFs were hydrolyzed with HCl-n-BuOH, cyanidin and delphinidin were detected. The partial structure of the MAFs was analyzed by pyrolysis-gas chromatography-mass spectrometry, and nine compounds were identified. These results suggest that MAFs are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates with intermonomeric linkages of B-ring to B-ring and C-ring to A-ring.
We examined the effect of okara on the prevention of obesity in mice. A modified AIN-76 diet with a high fat content (14.1% of crude fat) was used as a basal diet. Male ICR mice were fed ad libitum with the basal diet or a dried okara-supplemented basal diet (10, 20, or 40%) for 10 weeks. The okara intake dose-dependently suppressed the development of body weight and epididymal white adipose tissue (EWAT), and prevented an increase of plasma lipids, including total cholesterol, LDL cholesterol, and non-esterified fatty acid. The okara intake also prevented steatosis in the liver. Real-time RT-PCR revealed that the okara intake induced down-regulation of the fatty acid synthetase gene and up-regulation of the cholesterol 7 alpha-hydroxylase (CYP7A1) gene in the liver. We also found that the okara intake caused a marked reduction in the expression of leptin and TNF-alpha genes in EWAT. Our results suggest that okara is beneficial in preventing obesity.
Focusing on a functional component of Momordica charantia, saponin, we investigated its effects on serum glucose and neutral fat levels. Saponin was extracted as a butanol-soluble fraction (saponin fraction) from hot blast-dried Momordica charantia powder. The disaccharidase-inhibitory activity and the pancreatic lipase-inhibitory activity of the saponin fraction were measured, and in vivo sugar- and lipid-loading tests were performed. The saponin fraction inhibited disaccharidase activity and elevation of the blood glucose level after sucrose loading. The fraction also markedly inhibited pancreatic lipase activity and elevation of the serum neutral fat level after corn oil loading. Based on these findings, the main active component related to the anti-diabetic effect of Momordica charantia is present in the butanol fraction, and it may be saponin. The blood glucose and serum neutral fat-lowering effects of Momordica charantia were closely associated with its inhibitory activity against disaccharidase and pancreatic lipase.
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC–MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable –OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.
Mono-, di-, and trilinoleoyl glycerols were diluted with 1-undecanol or hexadecane to produce specific concentrations, and their oxidation processes were measured at 65 °C at 12% relative humidity. The rate constants for oxidation of the linoleoyl residue were proportional to the concentration for all substrates. This fact suggests that no intramolecular radical chain reaction between the linoleoyl residues occurred.
The effect of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) on the gene expression of Na+–K+–2Cl− co-transporter 1 (NKCC1) in intestinal epithelial cells was evaluated by using rats. The NKCC1 gene in ileal epithelial cells was significantly up-regulated by the oral administration of EC-12. The results of this study suggest in vivo that EC-12 had the potential to stimulate intestinal NKCC1 expression.
Inducing expression of the cholesterol-catabolizing enzyme cholesterol 7α-hydroxylase (CYP7A1) in the liver can be an effective strategy in preventing hypercholesterolemia and atherosclerosis. We used HepG2 cells to investigate the effects of 1 mM dipeptides having a C-terminal lysine group on the CYP7A1 mRNA level. We found that the dipeptides Asp-Lys, Glu-Lys, and Trp-Lys significantly increased the CYP7A1 mRNA level.
Sucrose monolauroyl esters were found to serve as substrates for cyclodextrin glucanotransferase (CGTase)-catalyzed transglucosidation reactions, affording new sucrose esters that have an additional 1-3 glucose residues on the pyranose ring of the sucrose moiety in the ester.
The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.
Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (Tm, the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.
Sludge production was reduced remarkably by reducing the dissolved oxygen supply to less than 1 mg/l in the conventional wastewater treatment procedure of a food-processing factory that produced 180 m3 of wastewater of biochemical oxygen demand (BOD) of about 1,000 mg/l daily. DNA was extracted from the sludge and subjected to PCR amplification. The PCR product was cloned into a plasmid and sequenced. Estimation of the resident bacterial distribution by 16S rDNA sequences before and after improvement of the system suggested a remarkable gradual change in the major bacterial population from Anaerolinaeceae (15.6%) to Comamonadaceae (52.3%), members of denitrifying bacteria of Proteobacteria. Although we did not directly confirm the ability of denitrification of the resulting sludge, a change in the major final electron acceptors from oxygen to nitrate might explain the reduction in sludge production in a conventional activated sludge process when the oxygen supply was limitted.
We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.