Biomineralization is a process of mineral deposition by organisms. Calcium salts are the major component of various biominerals, calcium carbonate being the predominant type in aquatic organisms. The mechanism of biomineralization has been conventionally analyzed by microscopic observation. The findings obtained suggest that minute amounts of organic matrices in biominerals play a key role in biomineralization. We first introduced the methodology of bioactive compound chemistry into this research field. Using various biominerals, such as the exoskeleton and gastroliths of the crayfish, the otoliths and scales of fish, the coccoliths of coccolithophores, bivalve shells, and coral skeleton, a range of organic matrices were purified by simple functional assays, and their chemical structures were determined. The function of each matrix component was estimated by its ability to interact with calcium carbonate and by in vitro crystallization, immunological localization, and site-specific and temporal expression of the encoding genes in the case of proteins and peptides, among other compounds. It was found that there was almost no similarity in chemical structure among organic matrices from various biominerals, but similarity in function was observed, and that made possible the functional classification of organic matrices.

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Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

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Highly sensitive detection of small peptides at the pM level was achieved by liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) in combination with the 2,4,6-trinitrobenzene sulfonate (TNBS) conversion technique. Six di-peptides having Tyr at the C-terminal (i.e., Gly-Tyr, Val-Tyr, Met-Tyr, Glu-Tyr, Lys-Tyr and His-Tyr) were subjected to the TNBS-MRM analysis in this study. The TNBS conversion conditions of pH 8.0, 30 °C and 60-min incubation enabled the di-peptides to be successfully converted to a trinitrophenyl (TNP) form with the mass increment of +212 Da. The proposed TNBS-MRM method enabled di-peptide detection that was highly improved by a factor of 3–55 in signal-to-noise ratio due to increased hydrophobicity by the induced TNP moiety. The method also permitted highly sensitive detection of di-peptides with a detection limit of >54 pM (>1.35 fmol/injection), achieving high reproducibility (<5% coefficient of variation) and rapidity (<30 min) by LC-MRM-MS/MS.

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The new lignan glucoside, acutumoside (1), was isolated from Sinomenium acutum rhizomes together with nine known compounds (2–10). The structure of 1 was elucidated on the basis of extensive spectroscopic analyses, including two-dimensional nuclear magnetic resonance and chemical reactions. Compounds 2, 7, 8, and 10 displayed potential antiproliferative activity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines, while compound 1 showed weak activity against these human tumor cells.

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L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

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The metabolic syndrome creates risk factors for coronary heart disease, diabetes, fatty liver, obesity and several cancers. Our group has already reported that the essential oil from leaves of Pinus koraiensis SIEB (EOPK) exerted antihyperlipidemic effects by upregulating the low-density lipoprotein receptor and inhibiting acyl-coenzyme A, cholesterol acyltransferases. We evaluated in the current study the anti-diabetic effects of EOPK on mice with streptozotocin (STZ)-induced type I diabetes and on HIT-T15 pancreatic β cells. EOPK significantly protected HIT-T15 cells from STZ-induced cytotoxicity and reduced the blood glucose level in STZ-induced diabetic mice when compared with the untreated control. EOPK consistently and significantly suppressed the α-amylase activity in a dose-dependent manner and enhanced the expression of insulin at the mRNA level in STZ-treated HIT-T15 cells, while the expression of insulin was attenuated. EOPK also significantly abrogated the population of reactive oxygen species when compared to the untreated control in STZ-treated HIT-T15 cells. Furthermore, EOPK significantly reduce nitric oxide production, suppressed the phosphorylation of endothelial nitric oxide (NO) synthase and suppressed the production of vascular endothelial growth factor (VEGF) in STZ-treated HIT-T15 cells, implying its potential application to diabetic retinopathy. Overall, our findings suggest that EOPK had hypoglycemic potential by inhibiting reactive oxygene species (ROS), endothelial NO synthase (eNOS) and VEGF in STZ-treated mice and HIT-T15 pancreatic β cells as a potent anti-diabetic agent.

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Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G₁, but the mechanism by which cells restart from G₁ delay remains elusive. We found that Whi3, a negative regulator of G₁ cyclin, counteracted Hog1 in the restart from G₁ delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G₁ delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G₁ arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G₁ arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.

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4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.

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Analysis of microarray data obtained by comparing gene expression between 2-week-old infant and 7-week-old mature SD rat testes revealed novel targets involved in tumor suppression. Reverse-transcription polymerase chain reaction and Northern blotting indicated that Tusc3 gene expression was upregulated in the normal maturing testis and prostate and other organs such as the cerebrum and ovary. Tumor suppressor candidate 3 protein expression was detected in these same organs at a size of about 40 kDa, in accord with the predicted molecular size. In situ hybridization and immunohistochemistry showed that mRNA and protein localization were prevalent in the testis spermatocytes and interstitial cells such as the Leydig cells, as well as prostate epithelial cells. These data suggest that TUSC3 is deeply involved in spermatogenesis in the testis, inducing sperm differentiation and maturation, and plays a role in normal prostate development and tumor suppression.

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By systematic analysis of high-throughput sequencing datasets from the human genome, we found that protein-coding genes have a specific chromatin structure near transcription termination sites relative to non-coding genes, one related to polyadenylation. Nucleosome was depleted near the site of cleavage/polyadenylation (polyA site) regardless of its relative position in the gene. DNA sequence plays an improtant role in nucleosome distribution, and conservative sequence elements and the protein binding to them are major determinants in causing nucleosome depletion near polyA sites. Furthermore, nucleosome occupancy was regulated by gene transcription and RNA polymerase II (RNAPII) occupancy. Our results reveal influences on nucleosome occupancy near polyadenylation sites and constitute evidence indicating that nucleosome distribution regulates 3′ end processing of protein-coding genes.

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We isolated and characterized two cDNA clone encoding warm-temperature acclimation-associated 65-kDa proteins (PoWap65-1 and PoWap65-2) from the olive flounder, Paralichthys olivaceus. The deduced amino acid sequences of PoWap65s showed overall identities of 33–73% with other fish Wap65 and mammalian hemopexin-like proteins. The 5′-flanking regions of both PoWap65-encoding genes contained various putative transcriptional elements. While PoWap65-1 and PoWap65-2 were structurally similar, they exhibited highly differential patterns of expression. PoWap65-1 was expressed only in the liver, whereas PoWap65-2 transcripts were detected in a wide range of tissues. The accumulation of PoWap65s mRNA was expressed differentially during development. Expression of them in warm temperatures also differed in flounder embryonic cells. PoWap65-1 was upregulated by temperature stimulation whereas PoWap65-2 was not detected. PoWap65s were highly regulated by Edwardsiella tarda infection and hypoxia. Pathogen challenge induced PoWap65-2 expression in the liver whereas PoWap65-1 was downregulated. Hypoxia induced the expression of both PoWap65s in the liver of juvenile fish.

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Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

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Limited and extensive proteolysis occur when β-conglycinin β homo-trimer (β₃-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of β₃-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of β₃-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and β₃-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.

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Ultrasound-assisted extraction was employed to extract polysaccharide from Virginia (flue-cured) tobacco flowers. The orthogonal matrix method (L₉(3)⁴) was used to determine the optimal extraction conditions as to ultrasound power, extraction time, ratio of solvent to solid, and extraction temperature at 300 W, 4 min, 35 (mL/g), and 70 °C respectively. The crude extract was successively purified by chromatography, yielding two major polysaccharide fractions, termed Fr-I and Fr-II. Both fractions are heteropolysaccharides, mainly containing glucose, mannose, and allose with an a-configuration. Thermo gravimetric analysis (TGA) indicated that the degradation temperatures (Td) of Fr-I and Fr-II were 185 °C and 190 °C respectively. The preliminary antioxidant activity test in vitro showed both fractions could potentialize the scavenging effect on hydroxyl and DPPH radicals in a dose-dependent manner. In conclusion, the two polysaccharides may be useful as naturally potential antioxidant agents for application in food and medicinal fields.

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For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.

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Cryptoquinone, an abietane-type diterpene para-quinone from the bark of Cryptomeria japonica has antifungal and cytotoxic activities, but its biological actions are largely unknown. In this study, we found that para-hydroquinone derivatives inhibited adipocyte differentiation. The actions might have been mediated, at least in part, by activation of the antioxidant-response element induction of phase 2 enzymes and increases in total glutathione.

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One of the most cost-effective methods of producing industrial enzymes is by the use of transgenic plants. We demonstrated successful high-level expression of a hyperthermostable archaeal β-1,4-endoglucanase in mature tobacco leaves by transformation of chloroplasts by homologous recombination. The active recombinant enzyme was readily recovered not only from fresh but also from dried leaves.

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We found that calcitonin gene-related peptide (CGRP) enhanced the expression of levels of serum amyloid A (SAA) and interleukin-6 (IL-6) in HepG2. In addition, CGRP-induced SAA1/2 mRNA expression was blocked by an anti-IL-6 neutralizing antibody in HepG2. These results suggest that CGRP promotes SAA synthesis through activation of IL-6 in human hepatocytes.

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Solitary chemosensory cells in the non-neuronal epithelium of the anterior nasal cavity have bitter taste cell-like molecular characteristics and are involved in the detection of noxious substances. Here, we demonstrate that Pou2f3/Skn-1a, which is necessary for generation of sweet, umami, and bitter taste cells, is also necessary for the generation or differentiation of solitary chemosensory cells.

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Using seven varieties of roasted sesame seeds, we examined the relationship between the fracture properties and the internal structure of roasted seeds. Analysis of fracture properties revealed significant differences across the samples in terms of fracture force and fracture strain at the biggest fracture point, and in the number of small fractures (p<0.05). Analysis of the cross-sectional structure revealed significant differences across the samples in the size of the void at the center of the sesame seed and in the overall thickness of the seed (p<0.05). Strong negative correlations were found for the center-void-to-seed thickness ratio against fracture force and fracture strain at the biggest fracture point. Moreover, fracture properties and cross-sectional structure were strongly correlated with sensory evaluations of crispness. Hence we concluded that the presence of a void in the central area of a seed decreases the necessary fracture force, resulting in a crispy texture.

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Proanthocyanidin (a persimmon-peel extract) is known to have potent antioxidative effects, but its protective action specifically against cellular damage has not been fully explored. In this work, we investigated the protective property of proanthocyanidin against cellular oxidative stress with an experimental model, H₂O₂-exposed human diploid fibroblasts (HDFs). To investigate the proposed underlying beneficial actions of proanthocyanidin as to cellular injury induced by H₂O₂, several major biochemical parameters were determined, including estimation of total reactive species (RS) generation, antioxidant enzyme activities, reduced glutathione (GSH)/oxidized glulathione (GSSG) ratio, and mitochondrial membrane potential. The results indicate that proanthocyanidin reduced total RS generation while enhancing the activities of catalase and glutathione reductase and the GSH/GSSG ratio. Additionally, proanthocyanidin was found to protect against mitochondrial membrane damage in HDFs treated H₂O₂. Based on these results, we conclude that proanthocyanidin has strong protective effects against cellular damage to several key cellular functions by suppressing oxidative stress in H₂O₂-treated HDFs.

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There is little information on L-tryptophan→nicotinamide metabolism in mice. In the present study, we investigated the two important nutritional factors involved in metabolism L-tryptophan→nicotinamide; one is the amount of nicotinamide synthesized from L-tryptophan, and the other is the urine ratio (N¹-methyl-2-pyridone-5-carboxamide + N¹-methyl-4-pyridone-3-carboxamide)/N¹-methylnicotinamide. The order of the percentages of nicotinamide synthesized from L-tryptophan was as follows: CBA strain mice (conversion percentage 0.41%) < BALB strain mice (0.82%) < C57BL/6 strain mice (1.13%) < ICR strain mice (1.70%). Urinary excretion of quinolinic acid was correlated with urinary excretion of the sum of nicotinamide and its catabolites (p<0.0001). The urine sum, which reflects the conversion of L-tryptophan→nicotinamide, correlated well with the activity of 3-hydroxyanthranilic acid dioxygenase (p=0.040). A nutritional indicator, the urine ratio (N¹-methyl-2-pyridone-5-carboxamide + N¹-methyl-4-pyridone-3-carboxamide)/N¹-methylnicotinamide, was controlled by the activity of N¹-methyl-2-pyridone-5-carboxamide-forming N¹-methylnicotinamide oxidase.

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Rice stems were subjected to a subcritical fluid treatment at 230 °C, using ethanol or acetone at a dilution of 0–100% in water. The obtained extracts were determined for their yield, carbohydrate content, phenolic content, DPPH radical scavenging ability, and color. The highest yield and carbohydrate content were achieved with the subcritical 20% (v/v) organic solvent, while the highest phenolic content was obtained with subcritical 80% (v/v) acetone. The highest radical scavenging ability was achieved with subcritical 60% (v/v) ethanol and 80% (v/v) acetone. The lightness of the extracts obtained with subcritical ethanol and acetone was negatively correlation with their radical scavenging ability (R=−0.85). The relationship between the lightness and phenolic content of the extracts was not significant, suggesting that other substances in the extract could also possess radical scavenging ability.

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We investigated the effects of a Eupatorium chinense var. simplicifolium (EUC) root extract on muscle disorders and explored the underlying mechanism for oxidative stress-induced C₂C₁₂ myoblast damage. An EUC pre-treatment reduced the decreased cell viability after an H₂O₂ treatment. The heat shock protein (HSP) 70 level increased, and the phosphorylation of Jun amino-terminal kinases (JNKs) decreased in the EUC-pre-treated C₂C₁₂ myoblasts. The results of the present study demonstrate the potential benefit of a herbal medicine in treating oxidative stress-related muscle disorders.

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L-Arabinose is a useful sugar in the food industry. We demonstrate here simple methods for refining arabinan polysaccharides by alcohol extraction from prune, Prunus domestica L., as a source of L-arabinose. Alcohol-soluble polysaccharides were purified from a solution of prune extracted by 80% ethanol. After fractionating the polysaccharides by ion-exchange chromatography, arabinans were identified as mainly constituted by (1→5)-linked arabinofuranosyl units.

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The administration of an ethanolic extract (RCE) from Rubus coreanus significantly reduced the body weight and epididymal fat tissue of mice under conditions of a high-fat diet (HFD) and exercise. The mice also displayed enhanced muscular carnitine palmitoyltransferase 1 (CPT1) expression and increased superoxide dismutase and glutathione levels. These results suggest that RCE exerted an anti-obesity effect by up-regulating CPT1 and elevating the level of antioxidants.

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To characterize novel variations of exopolysaccharides (EPSs) produced by dairy strains of Lactococcus lactis subsp. lactis and subsp. cremoris, the EPSs of five dairy strains of L. lactis were purified. Sugar composition analysis showed two novel EPSs produced by strains of L. lactis subsp. lactis. One strain produced EPS lacking galactose, and the other produced EPS containing fucose. Among the eps gene clusters of these strains, the highly conserved epsD and its neighboring epsE were sequenced. Sequence and PCR analysis revealed that epsE genes were strain-specific. By Southern blot analysis using epsD, the eps gene cluster in each strain was found to locate to the chromosome or a very large plasmid. This is the first report on the identification of two novel EPSs in L. lactis subsp. lactis. The strains can be detected among other strains by using epsE genes specific to them.

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Here we examined the involvement of Mycoplasma hominis in the formation of biofilms by uropathogenic Escherichia coli (UPEC) strain CFT073. Initially, we thought that M. hominis does not affect the fitness of UPEC, including the growth and production of signaling molecules, such as autoinducer-2 and indole. We found, however, that the presence of M. hominis significantly decreased the degree of biofilm formation by UPEC CFT073 (approximately a 60% reduction for 10⁵ ccu/mL of M. hominis as compared with UPEC alone). We also found that it had a slight effect in inhibiting the attachment and cytotoxicity of UPEC CFT073. These findings are specific to these UPEC strains rather than to enterohemorrhagic E. coli (EHEC) strains, found in normal intestinal flora. In addition, we performed whole-transcriptome profiling and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. This indicated that the PhoPQ system and the anti-termination protein (encoded by ybcQ) were involved in the reduction of biofilm formation by M. hominis (corroborated by qRT-PCR). Furthermore, our results indicate that M. hominis raises the degree of transcription of toxin genes, including hha and pasT. Hence, we suggest a possible role of M. hominis in affecting the formation of biofilms by UPEC in the urinary tract.

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The strain of Bacillus subtilis 168 used in laboratories in Japan was distributed in the 1990s when the sequencing consortium commenced operations. After 20 years of use of B. subtilis 168 in many laboratories, observations of variations in growth phenotypes have been reported. In this study, to uncover laboratory-specific variations of B. subtilis 168 strains in Japan, we re-sequenced 11 B. subtilis 168 strains from nine laboratories and analyzed how their genomes differed. We found that the 168 strains from different laboratories differed by 1–7 variations. These variations might have been caused by differences in storage conditions in the laboratories or differences among colonies of the original stock. Based on our results, researchers ought to understand the genetic differences among wild-type (parental) strains in different laboratories and the reference strain by re-sequencing analysis, and ought to pay more attention to the management of laboratory strains.

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The processing of archetypal Japanese sushi involves microbial fermentation. The traditional sushi kaburazushi, introduced in the middle ages, is made by fermenting salted yellow tail, salted turnip, and malted rice, and is distinguished from the ancient sushi narezushi, made from fish and boiled rice. In this study, we examined changes in the microbial population during kaburazushi fermentation by pyrosequencing the 16S ribosomal RNA genes (rDNA) of the organisms in the fermentation medium. Ribosomal Database Project Classifier analysis identified 31 genera, among which Lactobacillus drastically increased during fermentation (150-fold increment over 8 d), while the relative populations of the other gram-positive bacteria (Staphylococcus and Bacillus) decreased. Basic Local Alignment Search Tool analysis revealed the dominant species to be L. sakei. This organism constituted approximately 90% of Lactobacillus and 79% of total microbiota. The taxonomic diversity and species richness (assayed by Shannon-Weiner Index and Chao 1, respectively) were not significantly different between middle-ages kaburazushi and ancient narezushi. Both types were characterized by the preferential growth of Lactobacillales.

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We developed an efficient protocol that makes possible the practical use of Agrobacterium to transform the yeast-like conidia of Tremella fuciformis. Mechanical wounding of fungal cells prior to co-cultivation with Agrobacterium cells greatly improved transformation efficiency.

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The Japanese high-quality sake Daiginjo-shu is made from highly polished rice (polishing ratio, less than 50%). Here we showed that the sake rice Koshitanrei (KOS) has an excellent polishing property. Rice grains of KOS had the same lined white-core region as the sake rice Yamadanishiki (YAM). The grain rigidity/hardness of KOS was higher than that of the sake rice Gohyakumangoku (GOM). The loss ratio of KOS after high polishing by an industrial polishing machine was lower than that of GOM. Further, a clear taste of sake produced from KOS was confirmed by sensory evaluation.

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Benzylisoquinoline alkaloids (BIAs) are pharmaceutically important compounds. We have previously devised a reticuline (BIA) production method from dopamine by using Escherichia coli; however, its productivity was relatively low (33 µM, 11 mg/L). We report here, by fine-tuning the method, higher reticuline productivity of 165 µM (54 mg/L), increasing the conversion efficiency by 8-fold. These results are important for developing an efficient route to fermentative reticuline production.

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Batch, fed-batch, and continuous fermentation was used in the processing of methyl orange decolorization using growing cells of Lactobacillus casei TISTR 1500. This report presents the optimal conditions for methyl orange decolorization by the strain TISTR 1500 in modified MRS via a central composite design (CCD) experiment. In particular, the highest decolorization efficiencies were obtained with 13.41 g/L of meat extract, and with 10.89 g/L of yeast extract at pH 6.88 at 35 °C. Under the optimal conditions, the rate of decolorization increased to 322% of that obtained for un-optimized MRS medium. The high concentration of methyl orange (5 g/L) was completely degraded within 9 h in batch fermentation. The total methyl orange load with 8.075 g/L was also decolorized in fed-batch fermentation within 13 h, and the biomass of the strain dramatically decreased after an incubation time of 8 h due to a shortage of sucrose. In the continuous system with a dye-loading rate of 600 mg/L/h and a total of loaded azo dye of 7.2 g/L, high efficiency of methyl orange removal was significantly high, at 98%.

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In Arabidopsis thaliana, the aldo-keto reductase (AKR) family includes four enzymes (The AKR4C subfamily: AKR4C8, AKR4C9, AKR4C10, and AKR4C11). AKR4C8 and AKR4C9 might detoxify sugar-derived reactive carbonyls (RCs). We analyzed AKR4C10 and AKR4C11, and compared the enzymatic functions of the four enzymes. Modeling of protein structures based on the known structure of AKR4C9 found an (α/β)₈-barrel motif in all four enzymes. Loop structures (A, B, and C) which determine substrate specificity, differed among the four. Both AKR4C10 and AKR4C11 reduced methylglyoxal. AKR4C10 reduced triose phosphates, dihydroxyacetone phosphate (DHAP), and glyceraldehydes 3-phosphate (GAP), the most efficiently of all the AKR4Cs. Acrolein, a lipid-derived RC, inactivated the four enzymes to different degrees. Expression of the AKR4C genes was induced under high-[CO₂] and high light, when photosynthesis was enhanced and photosynthates accumulated in the cells. These results suggest that the AKR4C subfamily contributes to the detoxification of sugar-derived RCs in plants.

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