Atherosclerosis is a multifactorial, long-lasting process in humans. Accordingly, animal models in which more rapid changes occur can be useful for the study of this process. Among such models are apolipoprotein E-deficient (apoE−/−) mice, which give insight into the human process. ApoE−/− mice show impaired clearing of plasma lipoproteins and develop atherosclerosis in a short time, and hence they are an excellent model in which to assess the impact of dietary factors. This review considers lipid metabolism and inflammation as well as nutritional constituents affecting atherosclerosis, with reference to apoE−/− mice, and discusses the mechanisms through which they act.
Zinc plays essential roles in the early secretory pathway for a number of secretory, membrane-bound, and endosome/lysosome-resident enzymes. It enables the enzymes to fold properly and become functional, by binding as a structural or catalytic component. Moreover, zinc secreted from the secretory vesicles/granules into the extracellular space has a pivotal role as a signaling molecule for various physiological functions. Zinc transporters of the Slc30a/ZnT and Slc39a/Zip families play crucial roles in these functions, mediating zinc influx to and efflux from the lumen of the secretory pathway, constitutively or in a cell-specific manner. This paper reviews current knowledge of the ways these two zinc transporters perform these tasks by manipulating zinc homeostasis in the secretory pathway. Recent questions concerning zinc released into the cytoplasm from the secretory pathway, which then functions as an intracellular signaling molecule, are also briefly reviewed, emphasizing zinc transporter functions.
The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed.
Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase Cδ, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.
A new steroidal saponin was isolated from Ophiopogon japonicus. This saponin possesses a modification by 2-hydroxy-3-methylvalerylation of the hydroxyl group at C-4′ of the sugar, linked to C-1 of the aglycone. It exhibited significant neuritogenic activity for PC12 cells. The structure-activity relationship revealed the aglycone, rather than the sugar moieties and acylation, to be important for the neuritogenic activity.
A novel (R)-specific alcohol dehydrogenase (AFPDH) produced by Candida maris IFO10003 was purified to homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl, and Phenyl-Toyopearl, and characterized. The relative molecular mass of the native enzyme was found to be 59,900 by gel filtration, and that of the subunit was estimated to be 28,900 on SDS-polyacrylamide gel electrophoresis. These results suggest that the enzyme is a homodimer. It required NADH as a cofactor and reduced various kinds of carbonyl compounds, including ketones and aldehydes. AFPDH reduced acetylpyridine derivatives, β-keto esters, and some ketone compounds with high enantioselectivity. This is the first report of an NADH-dependent, highly enantioselective (R)-specific alcohol dehydrogenase isolated from a yeast. AFPDH is a very useful enzyme for the preparation of various kinds of chiral alcohols.
Taste buds contain three types of taste cells. Each type can respond to taste stimulation, and type II and III taste cells are electrically excitable. However, there are differences between the properties of type II and III taste cells. In this study, we found that Fxyd6, an Na,K-ATPase regulator gene, is expressed in type II taste cells in the taste buds of mice. Double-labeled in situ hybridization analysis showed that Fxyd6 was coexpressed with transient receptor potential cation channel, subfamily M, member 5 (Trpm5), a critical component of the sweet, bitter, and umami taste signal transduction pathways and that it was specifically expressed in type II taste cells. We also found that taste cells frequently coexpressed Fxyd6 and Na,K-ATPase β1. These results indicate the presence of an inherent mechanism that regulated transmembrane Na+ dynamics in type II taste cells.
In this study, we investigated the effects of a petroleum ether fraction of Artemisia sacrorum Ledeb. (Compositae) (PEASL) on glucose production through AMP-activated protein kinase (AMPK) activation in human HepG2 cells. PEASL significantly inhibited glucose production in a concentration-dependent manner, and this effect was reversed in the presence of compound C, a selective AMPK inhibitor. PEASL markedly induced the phosphorylation of AMPK and downstream acetyl-CoA carboxylase (ACC) in a time- and concentration-dependent manner. In addition, it markedly increased the phosphorylations of glycogen synthase kinase 3β (GSK3β) in a concentration-dependent manner. In contrast, cAMP reponse element binding protein (CREB), a key transcription factor for gluconeogenic enzyme phosphorylation, decreased in a concentration-dependent manner. PEASL downregulated the gluconeogenesis gene expression of peroxisome proliferation activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in a concentration-dependent manner. In addition, the gene expression of orphan nuclear receptor small heterodimer partner (SHP) increased, also in a concentration-dependent manner. These effects were also abolished by pretreatment with compound C, an AMPK inhibitor. This indicates that PEASL inhibited glucose production via the AMPK-GSK-CREB pathway in HepG2 cells, and these effects appeared to be capable of revealing anti-diabetic mechanism of PEASL in HepG2 cells.
The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.
We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.
Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T5 of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine–SDS–PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.
To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105–115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.
The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.
Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.
Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.
Selenite (SeO32−) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/autoradiography using 75Se-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).
The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.
A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.
We isolated a homolog of cathepsin L from a cDNA library of the olive flounder liver. The flounder cathepsin L1 transcript consisted of 1,221 bp that encoded a polypeptide of 334 amino acids. The overall identity between flounder cathepsin L1 and other cathepsin Ls was 50–64%, and flounder cathepsin L1 contained the highly conserved ERFNIN-motif. A phylogenetic tree indicated that flounder cathepsin L1 is in the same monophyletic group as zebrafish cathepsin Lc. RT-PCR analysis revealed that cathepsin L1 transcripts were expressed only in the liver. They were detected from 28 d post-hatching. Under starvation conditions, cathepsin L1 expression was decreased at 30 d.
-Gingerol is an active component of ginger that shows antipyretic and anti-inflammation activities. To find a novel skin-whitening agent, the melanogeneis inhibitory effects and action mechanisms of -gingerol were investigated. In the present study, the effects of -gingerol on mushroom tyrosinase, tyrosinase activity, and melanin content were determined spectrophotometrically, and the expression of tyrosinase and related proteins in B16F10 murine melanoma cells was evaluated by Western blotting. Furthermore, a possible signaling pathway involved in -gingerolmediated depigmentation was investigated by means of specific inhibitors. The results revealed that -gingerol (25–100 μM) effectively suppresses murine tyrosinase activity and decreases the amount of melanin in a dose-dependent manner. Additionally, it also effectively decreased the intracellular reactive oxygen species (ROS) level in a dose-dependent pattern in the same dose range. Our results indicate that -gingerol inhibits melanogenesis of B16F10 melanoma and can function as a good skinwhitening agent.
Guava leaves were classified and the free radical scavenging activity (FRSA) evaluated according to different harvest times by using the 1H-NMR-based metabolomic technique. A principal component analysis (PCA) of 1H-NMR data from the guava leaves provided clear clusters according to the harvesting time. A partial least squares (PLS) analysis indicated a correlation between the metabolic profile and FRSA. FRSA levels of the guava leaves harvested during May and August were high, and those leaves contained higher amounts of 3-hydroxybutyric acid, acetic acid, glutamic acid, asparagine, citric acid, malonic acid, trans-aconitic acid, ascorbic acid, maleic acid, cis-aconitic acid, epicatechin, protocatechuic acid, and xanthine than the leaves harvested during October and December. Epicatechin and protocatechuic acid among those compounds seem to have enhanced FRSA of the guava leaf samples harvested in May and August. A PLS regression model was established to predict guava leaf FRSA at different harvesting times by using a 1H-NMR data set. The predictability of the PLS model was then tested by internal and external validation. The results of this study indicate that 1H-NMR-based metabolomic data could usefully characterize guava leaves according to their time of harvesting.
We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.
A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS–PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A4 hydrolase (LTA4H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.
Oxygenation of lipids during the processing soybeans affects the flavor properties of soy products. We prepared tofu under anaerobic conditions and then evaluated its sensory properties and the compositions of volatiles and oxidized lipids. Anaerobic processing resulted in tofu with less intense richness (kokumi) concomitant with reductions in the amounts of oxidized lipids and volatile compounds.
This study investigates whether there is a predominant Staphylococcus aureus strain in retail foods and healthy human hands, and examines the relationship between pulsed-field gel electrophoresis (PFGE) banding patterns and the S. aureus characteristics of staphylococcal enterotoxin (SE) type, coagulase type, and β-lactamase activity. Ninety-four strains of S. aureus isolated from retail foods and healthy human hands were analyzed by PFGE. Several strains isolated from the same shop or a chain store showed identical patterns, indicating that the origins of these strains were identical. After excluding these strains showing identical patterns, 54 strains were used for the PFGE analysis. No spread of a particular clone in the environment surrounding the food was apparent. The PFGE analysis of these 54 strains was classified in 6 lineages (L1–L6). There was no relationship between the PFGE banding pattern and coagulase type or SE type. Eleven (84.6%) of the 13 isolates in PFGE banding pattern L5 did not produce β-lactamase, suggesting that the production of β-lactamase influenced a specific PFGE banding pattern.
The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.
Scanning electron micrographs show that perilla globulin gel had a finer network structure than sesame α-globulin gel. The effects of various reagents on the gel formation and solubility of perilla and sesame gels were compared. The contribution of disulfide bonds to the formation and stability of perilla gel was greater than to sesame gel, despite having the same subunit structure.
Coffee “silverskin” (CS) is a by-product of the roasting procedure for coffee beans. A CS extract (CS-ext) was found to have a high inhibitory effect against hyaluronidase. It seems that the higher-molecular-weight substances in CS-ext contributed most to the hyaluronidase inhibition, while acidic polysaccharides mainly composed of uronic acid played a major role in this hyaluronidase inhibition by CS-ext.
In this study, we investigated the relationship between MgSO4 and luminescence in Vibrio fischeri under nutrient-starved conditions. When V. fischeri was cultured in an artificial seawater medium, the luminescence intensity was low relative to that observed under normal growth conditions. It decreased during the initial 14 h, and then increased slightly at 24 h. This regulation of luminescence was not dependent on the quorum-sensing mechanism, because the cell densities had not reached a critical threshold concentration. Under MgSO4-starved conditions, luminescence was not fully induced at 14 h, and decreased at 24 h. In contrast, induction of luminescence occurred under MgSO4-supplemented conditions, but MgSO4 alone was insufficient to induce luminescence, and required NaHCO3 or KCl. These results suggest that the luminescence of V. fischeri is controlled by an exogenous sulfur source under nutrient-starved conditions. In addition, they indicate that the induction of sulfur-dependent luminescence is regulated by the NaHCO3 or KCl concentration.
Schizosaccharomyces pombe treated with valproic acid died with apoptotic markers such as DNA fragmentation, loss of a mitochondrial electrochemial gradient and chromatin condensation, independently of metacaspase, a yeast homolog of metazoan caspase. Sensitivity to valproic acid was strongly dependent on growth phase. Cells in a later growth phase were much more sensitive to valproic acid than those in an earlier one. Altering the pH of the medium with HCl and with NaOH also caused remarkable changes in sensitivity. Cells in an acidic medium were more sensitive to valproic acid. This pH-dependent change in sensitivity did not require de novo protein synthesis, and a change in pH 60 min after the administration of valproic acid affected sensitivity. These results suggest that the intracellular cell death process was susceptible to extracellular pH. Although a sir2 mutant of Saccharomyces cerevisiae has been reported to be resistant to valproic acid, mutations in sir2 did not affect the sensitivity to valproic acid of S. pombe.
A crude endo-xylanase produced by Aspergillus niger BCC14405 was investigated for its potential in pre-bleaching of chemical pulp from eucalyptus. The optimal fermentation conditions on the basis of optimization using response surface methodology included cultivation in a complex medium comprising wheat bran, rice bran, and soybean meal supplemented with yeast extract, glucose, peptone, and lactose with a starting pH of 6.0 for 7 d. This resulted in production of 89.5 IU/mL of xylanase with minor cellulase activity. Proteomic analysis using LC/MS/MS revealed that the crude enzyme was a composite of hemicellulolytic enzymes, including endo-β-1,4-xylanase and other hemicellulolytic enzymes attacking arabinoxylan and mannan. Pretreatment of the pulp at a xylanase dosage of 10 IU/g increased the brightness ceiling after the C-Eop-H bleaching step up to 3.0% using a chlorine charge with a C-factor of 0.16–0.20. Xylanase treatment also led to reduction in chlorine charge of at least 20%, with an acceptable brightness level. The enzyme pretreatment resulted in a slight increase in pulp viscosity, suggesting an increase in relative cellulose content. The crude enzyme was potent in the enzyme-aided beaching of chemical pulp in an environmentally friendly pulping process.
Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.
One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP≧4 than KP from Thermoanaerobacter brockii ATCC35047.
An understanding of the cadmium (Cd) transport system in rice can serve as a basis for coping with Cd-related problems in rice and human health. To identify a new gene involved in Cd transport in rice, we screened our yeast library, expressing 140 kinds of rice ATP binding cassette (ABC)-type transporters. From the screening, we found that OsABCG43/PDR5 conferred high Cd tolerance on yeast. The Cd concentration of yeast carrying OsABCG43 was similar to that of the vector control. The OsABCG43 transcript was detected both in shoots and roots. Accumulation of it was elevated by Cd treatment in the roots but not in the shoots. This study indicates that OsABCG43 is a Cd inducible-transporter gene capable of conferring Cd tolerance on yeast.