Carbazole degradation by several bacterial strains, including Pseudomonas resinovorans CA10, has been investigated over the last two decades. As the initial reaction in degradation pathways, carbazole is commonly oxygenated at angular (C9a) and adjacent (C1) carbons as two hydroxyl groups in a cis configuration. This type of dioxygenation is termed “angular dioxygenation,” and is catalyzed by carbazole 1,9a-dioxygenase (CARDO), consisting of terminal oxygenase, ferredoxin, and ferredoxin reductase components. The crystal structures of all components and the electron transfer complex between terminal oxygenase and ferredoxin indicate substrate recognition mechanisms suitable for angular dioxygenation and specific electron transfer among the three components. In contrast, the carbazole degradative car operon of CA10 is located on IncP-7 conjugative plasmid pCAR1. Together with conventional molecular genetic and biochemical investigations, recent genome sequencing and RNA mapping studies have clarified that transcriptional cross-regulation via nucleoid-associated proteins is established between pCAR1 and the host chromosome.
A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.
The Rho-associated serine-threonine protein kinase (ROCK) is a downstream effector of Rho GTPases that is frequently activated in the epithelial to mesenchymal transition (EMT) of human ovarian cancer cells. On the other hand, endothelin-1 (ET-1) and its receptor endothelin A receptor (ETAR) are overexpressed in primary and metastatic ovarian carcinoma, which suggests that ET-1 promotes tumor dissemination. Hence, two human ovarian carcinoma cell lines, SK-OV-3 and CaOV3, were chosen to study the effects of ET-1/ETAR and ROCK in promoting EMT of ovarian cancer cells. We found that ET-1 exposure induced EMT of SK-OV-3 and CaOV3 by monitoring cells morphology, enhanced fibronectin, and reduced E-cadherin protein. At the same time, ET-1/ETAR enhanced the level of transcription of SLUG a transcriptional repressor of E-cadherin. More importantly, a constitutively active mutant of ROCK enhanced the transcription of SLUG by stimulating SLUG promoter activity. Furthermore, ROCK inhibitor Y27632 reversed the increase in fibronectin induced by ET-1/ETAR. Our data suggest that ROCK cooperated with ET-1/ETAR to promote EMT of human ovarian carcinoma cells through upregulation of SLUG mRNA.
The gene encoding isocitrate lyase (ICL) from a nitrogen-fixing mesophilic bacterium, Azotobacter vinelandii strain IAM1078, was cloned, and the gene expression was examined. When sodium acetate or glucose was used as carbon source, similar growth was observed in this bacterium, but the ICL activity of cells grown with the former source was 43-hold higher than those with the latter. In addition, northern blot analysis revealed that expression of the ICL gene was induced by acetate. Based on a comparison of the amino acid sequences of the ICLs of various organisms, the ICL of this bacterium was found to be classifiable into subfamily 3, one of two phylogenetic groups of eubacteial ICLs. Replacement of the Ile504 in the ICL by Met, which is conserved in the corresponding position of cold-adapted ICLs of psychrophlic bacteria, resulted in decreased thermostability of activity, indicating that this amino acid residue is involved in thermal properties of this enzyme.
Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T50, of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0–55.5 °C, 4.7–5.2 °C higher than without sugar (50.3±0.1 °C). The T50 of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the kcat of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) is involved in numerous cellular responses. In this study, we investigated the protective effects of PDK1 gene expression against hypoxic conditions in cultured rat CMCs (rCMCs) and in a rat myocardial infarction (MI) model using the lentiviral vector (LeV) system. LeV-PDK1 transfer effectively reduced the apoptotic cell death caused by hypoxic injury as compared to LeV-GFP transfer in rCMCs the expression of survival proteins increased in the LeV-PDK1 group, whereas apoptosis signaling decreased in the rCMCs and in infarcted hearts treated with LeV-PDK1. LeV-PDK1 transfer also reduced apoptosis and infarct size and attenuated myocardial wall thinning and ventricular remodeling in a rat MI model. These findings suggest that PDK1 has a protective role in the injured ischemic myocardium via overexpression of the cell survival pathway in CMCs. Hence PDK1 can be used as a treatment strategy for myocardial salvage inin hypoxic injury.
Mouse G258 mutant stopped both cell growth and the synthesis of lipid-linked oligosaccharide at the Man3GlcNAc2-P-P-Dolichol at a restricted temperature with a single gene mutation. To clarify the lesion in the G258 mutant, we isolated human genomic DNA transformants of the G258 mutant, which recovered from both defects by way of cell hybridization with X-ray irradiated HeLa cells. We detected a common 1.3-kb product by inter-human specific sequence in the L1 (L1Hs) PCR in the transformants (Kataoka et al., Somat. Cell Mol. Genet., 24, 235–243 (1998)). In the present study, we screened a human mega yeast artificial chromosome (YAC) library by PCR with primers designed according to the 1.3-kb DNA, and selected YAC clone 923f5. Moreover, we found by spheroplast fusion that YAC clone 923f5 complemented both defects of the G258 mutant. Since the human counterpart of the yeast ALG11 gene is localized in the region, the G258 mutant might have a defect in the mouse ALG11 gene.
The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.
Coenzyme A (CoA) is an essential, ubiquitous cofactor in all biological systems, where it acts as the major acyl group carrier in various central metabolic reactions. Although much is known about CoA biosynthesis, it is unclear how the CoA pool is regulated the various cellular compartments. It has been found that the nucleoside diphosphates linked to some moiety X (Nudix) hydrolases, AtNUDX11 and 15, have pyrophosphohydrolase activity toward CoA and its derivatives. In this study we identified two alternatively spliced variants, AtNUDX15 and 15a, produced from the AtNUDX15 gene, and carried out comparative studies of the gene regulation, the kinetic parameters, and the intracellular localization of AtNUDX11, 15, and 15a. The present findings indicate that AtNUDX11 and AtNUDX15(a) function in the hydrolysis of malonyl-CoA in cytosol and succinyl-CoA in the mitochondria, respectively, suggesting their impact not only on CoA biosynthesis but also on various CoA-related pathways such as the TCA cycle.
Both the propeptide in the precursor carboxypeptidase Y (proCPY) and the mature CPY (mCPY)-specific endogenous inhibitor (IC) inhibit CPY activity. The N-terminal inhibitory reactive site of IC (the N-terminal seven amino acids of IC) binds to the substrate-binding site of mCPY and is essential for mCPY inhibition, but the mechanism of mCPY inhibition by the propeptide is poorly understood. In this study, sequence alignment between IC and proCPY indicated that a sequence similar to the N-terminal region of IC was present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of IC. In the presence of peptides identical to the N-terminus of IC and the C-terminus of the propeptide, CPY activity was competitively inhibited. The C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.
Antimicrobial peptides are effector molecules of the innate immunity of amphibians. Here, one antimicrobial peptide cDNA precursor, prepropalustrin-2CE3, from the tadpole of the Chinese brown frog Rana chensinensis was cloned. The coding sequence corresponding to the mature palustrin-2CE peptide was subcloned into pGEX-6p-1. The soluble GST-palustrin-2CE fusion protein was successfully expressed in the BL21(DE3)pLysS strain at 16 °C, and the proportion of the fusion protein reached 35%–39% of the total cellular protein. After removal of the GST-fusion tag, the purity of the palustrin-2CE obtained by Sephadex G50 chromatography was about 97%. Moreover, the purified palustrin-2CE displayed obviously inhibitory activities against the sensitive bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and multi-drug resistant S. aureus and E. coli. These findings suggest that the tadpole of the Chinese brown frog is a unique source of antimicrobial peptides and indicates the therapeutic potential of the palustrin-2CE peptide.
Endothelial progenitor cells (EPCs) are applied in the treatment of ischemic diseases. In ex vivo culture of human cord-blood derived EPCs, H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine, markedly increased the number of EPCs. It also induced EPC migration, stimulated the phosphorylation of AKT, and reduced the expression of p27 in the EPCs. Thus H1152 can be used effectively in ex vivo expansion of EPCs.
A gene coding for a major extracellular chitosanase was isolated from Aspergillus oryzae IAM2660. It had a multi-domain structure composed of a signal peptide, a catalytic domain, Thr- and Pro-rich linkers, and repeated peptides (the R3 domain) from the N-terminus. The R3 domain bound to insoluble powder chitosan, but it did not promote the hydrolysis rate of the chitosanase to any extent.
We attempted to inactivate endopolygaolacturonase from Stereum purpureum (EndoPG) IV of identical origin by linking the pro-sequence of S. purpureum Pro-EndoPG I to the C-terminus. The recombinant Pro-EndoPG IV, expressed in Escherichia coli, had no polygalacturonase (PG) activity, but activity was acquired after partial degradation of the pro-sequence with V8 protease, as was the case for Pro-EndoPG I. These results indicate that the pro-sequence of Pro-EndoPG I can suppress the PG activity of EndoPG IV.
A malfunction in the yeast HAC1 causes the unfolding-protein response in the endoplasmic reticulum, resulting in stress-sensitive and inositol auxotrophic phenotypes. Chaperonin-containing TCP1 (CCT) is necessary for the folding of actin and tubulin in the cytosol. The introduction of the truncated human CCT epsilon subunit into yeast cells of which hac1 was disrupted clearly suppressed not only its inositol auxotrophic phenotype but also its stress-sensitive phenotype.
Pyrrolysine-tRNAPyl complex is produced by pyrrolysyl-tRNA synthetase (PylRS). In this study, we investigated the substrate specificity of Desulfitobacterium hafnience PylRS. PylRS incorporated various L-lysine derivatives into tRNAPylin vitro. In addition, the PylRS/tRNAPyl pair introduced these lysine derivatives into the recombinant protein by the Escherichia coli expression system, indicating that this PylRS/tRNAPyl pair can be used in protein engineering technology.
Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.
We examined the effects of lactic acid fermentation of soymilk on the lipid profile and lipid metabolism-related gene expression in rat liver. Male Sprague-Dawley rats aged 7 weeks were fed a control diet (AIN-93), soymilk diet, or fermented soymilk diet for 1 week or 5 weeks. The hepatic triglyceride and cholesterol contents in the soymilk (SM) group and the fermented soymilk (FSM) group were significantly lower than those in the control group after 5 weeks, but these changes had not become apparent until after 1 week. The fatty acid synthesis-related genes were more markedly down-regulated after 1 week than after 5 weeks, whereas the cytochrome p450 family 7 subfamily a polypeptide 1 (CYP7al) gene related to cholesterol catabolism was more markedly up-regulated after 5 weeks than after 1 week. This up-regulation was higher in the FSM group than in the SM group. It is assumed that the bioactive components produced by lactic acid fermentation induced the up-regulation of CYP7a1.
High pressure was applied to recombinant human lactoferrin obtained from rice (rhLF) and its effect was evaluated on the structure and activity of the protein. Treatments of 400, 500, and 650 MPa for 15 min (20 °C), were applied to rhLF at 2 mg/mL in three iron-saturation forms. The structural characteristics of the treated proteins were analyzed by differential scanning calorimetry (DSC) and by fluorometric analysis, and immunoreactivity by ELISA. Iron retention and binding properties and antibacterial activity against Escherichia coli O157:H7 were also studied. The results obtained indicate that the treatments at 400 and 500 MPa did not greatly modifiy the conformation of lactoferrin, meanwhile treatment at 650 MPa affected in different degrees the three forms of rhLF. With respect to antibacterial activity, only apo rhLF showed antibacterial activity against E. coli, activity that was maintained after treatment at 400 MPa, while holo and AsIs rhLF did not inhibit the growth of E. coli.
We investigated the effects of water-soluble low-molecular-weight β-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans 1A1 strain black yeast (LMW-β-glucan) on mast cell-mediated anaphylactic reactions. Although it is known that LMW-β-glucan has anti-tumor, anti-metastatic and anti-stress effects, the roles of LMW-β-glucan in immediate-type allergic reactions have not been fully investigated. We examined whether LMW-β-glucan could inhibit mast cell degranulation and passive cutaneous anaphylaxis (PCA). LMW-β-glucan dose-dependently inhibited the degranulation of both rat basophilic leukemia (RBL-2H3) and cultured mast cells (CMCs) activated by calcium ionophore A23187 or IgE. However, LMW-β-glucan had no cytotoxicity towards RBL-2H3 cells and CMCs. Furthermore, orally administered LMW-β-glucan inhibited the IgE-induced PCA reaction in mice. These results show LMW-β-glucan to be a possible compound for the effective therapeutic treatment of allergic diseases.
The crude extract of peach leaves dose-dependently suppressed the postprandial elevation in the blood glucose level after an oral administration of soluble starch to mice. This study examines the mechanism for this suppressive effect in vivo. An oral carbohydrate-loading test on mice showed that the peach leaf extract suppressed the glucose-induced increase in the blood level of glucose, but without affecting the insulin level. An enteral soluble starch and glucose loading test on mice also showed that the crude extract (1,000 mg/kg) significantly suppressed the postprandial elevation of the blood glucose level and increased the amount of glucose that remained in the intestine to within the same range as that with phloridzin (500 mg/kg), a natural sodium-dependent glucose transporter (SGLT)-specific inhibitor. In contrast, the extract did not suppress the postprandial elevation of the blood triglyceride and cholesterol levels in mice, and did not affect the normal blood glucose level in a feeding test for 21 d. These results reveal that the extract of peach leaves suppressed the postprandial elevation of blood glucose level by inhibiting the absorption of glucose in the small intestine of mice.
Carnosic acid, a diterpene in rosemary, is considered to be beneficial in the prevention of chronic neurodegenerative diseases. Recently, it has been found that drugs with antiangiogenic activity lower the risk of neurodegenerative diseases. Thus it is of interest whether carnosic acid has antiangiogenic activity. In this study, carnosic acid suppressed microvessel outgrowth on ex vivo angiogenesis assay using a rat aortic ring at higher than 10 μM. The antiangiogenic effect of carnosic acid was found in angiogenesis models using human umbilical vein endothelial cells with regard to tube formation on reconstituted basement membrane, chemotaxis and proliferation. Although the carnosol in rosemary also suppressed angiogenesis, its effect was not more potent than that of carnosic acid in the ex vivo model. These results suggest that carnosic acid and rosemary extract can be useful in the prevention of disorders due to angiogenesis, and that their antiangiogenic effect can contribute to a neuroprotective effect.
Two dipeptides, glycyl-L-leucine (G-L) and L-leucyl-glycine (L-G), the concentrations of which were 10 mmol/L, were degraded in subcritical water in order to understand fully the phenomena occurring during treatment. Treatment was administered in a stainless steel tubular reactor, which was connected to an HPLC pump and immersed in an oil bath at 200–240 °C, with residence times of 10–180 s. When G-L and L-G were treated, L-G and G-L significantly formed, respectively, and then they gradually decreased at every temperature. Irrespective of the kind of substrate, ring formation occurred, and cyclo-(glycyl-L-leucine) was one of the final products. The reaction rate constants related to degradation were estimated under the assumption that all the reactions obeyed first-order kinetics, and the simulated results corresponded well with the experimental ones in every case.
Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The 13C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.
Marine plants have been reported to possess various pharmacological properties; however, there have been few reports on their neuropharmacological effects. Terrestrial plants have depressive effects on the central nervous system (CNS) because of their polyphenols which make them effective as anticonvulsants and sleep inducers. We investigated in this study the depressive effects of the polyphenol-rich brown seaweed, Ecklonia cava (EC), on CNS. An EC enzymatic extract (ECEE) showed significant anticonvulsive (>500 mg/kg) and sleep-inducing (>500 mg/kg) effects on the respective mice seizure induced by picrotoxin and on the mice sleep induced by pentobarbital. The phlorotannin-rich fraction (PTRF) from ECEE significantly potentiated the pentobarbital-induced sleep at >50 mg/kg. PTRF had binding activity to the gamma aminobutyric acid type A (GABAA)-benzodiazepine (BZD) receptors. The sleep-inducing effects of diazepam (DZP, a well-known GABAA-BZD agonist), ECEE, and PTRF were completely blocked by flumazenil, a well-known antagonist of GABAA-BZD receptors. These results imply that ECEE produced depressive effects on CNS by positive allosteric modulation of its phlorotannins on GABAA-BZD receptors like DZP. Our study proposes EC as a candidate for the effective treatment of neuropsychiatric disorders such as anxiety and insomnia.
Supplementation with lupeol (0.67 g·kg−1) of the AIN-93M-based diet fed for 7 weeks to stroke-prone spontaneously hypertensive rats caused significantly decreased blood pressure as compared with a control group. Urinary 8-hydroxy-2′-deoxyguanosine was significantly lower in the lupeol group. Finally, lupeol suppressed the hepatic mRNA expression levels of the genes involved in triglyceride and cholesterol synthesis.
Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N1-methyl-2-pyridone-5-carboxamide + N1-methyl-4-pyridone-3-carboxamide)/N1-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.
The methanol extract of Dypsis lutescens leaves showed inhibitory effects on lipase activity in vitro and on triglyceride accumulation in 3T3-L1 pre-adipocytes. Further experiments using the extract on mice demonstrated a suppressive effect on the postprandial elevation of blood triglyceride level and an anti-obesity effect on obese mice induced by a high-fat diet. D. lutescens will accordingly be useful for preventing obesity.
Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.
The antioxidative effect of fullerenes C60 and C70 was examined by measuring the inhibition of methyl linoleate (MeL) peroxidation in toluene initiated by 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). The fullerenes retarded the formation of MeL hydroperoxides and lowered the rate of propagation. The reaction rates of fullerenes with AMVN-derived peroxyl radicals were much higher than that of MeL. These results indicate that fullerenes can act as retarders of lipid peroxidation, though their activity is low compared with that of α-tocopherol.
The characteristics of a bacteriocin from Ruminococcus albus 7 and its potential as an antibiotic alternative were examined in this study. The addition of 3 μM 3-phenylpropanoic acid (PPA) and 0.2% Tween 80 to the culturing medium improved bacteriocin production by 2.5-fold. Native polyacrylamide gel electrophoresis of the antagonistically active gel filtration fraction established that the molecular weight of the R. albus 7 bacteriocin was approximately 36 kDa. The bacteriocin was sensitive to pepsin, protease, and pancreatin, and was inactivated by heating at 65 °C for 1 h. Simulating in vitro avian digestion decreased the antagonistic activity by 74.7%, but the addition of 1% bovin serum albumin restored 13% of the lost antagonistic activity. Following ion-exchange purification, the bacteriocin had sufficient antagonistic activity against five tested pathogenic strains, but the addition of a protectant is necessary for utilization of bacteriocin of R. albus 7 as an antibiotic alternative in animal feed.
Modern Japanese sushi is derived from an archetype, narezushi, which is made by the fermentation of salted fish with rice. Several studies have demonstrated that lactic acid bacteria are dominantly present in narezushi, but no studies have addressed how microbial composition changes during fermentation. In this study, we examined changes in the microbial population in aji (horse mackerel)-narezushi during fermentation by pyrosequencing the 16S ribosomal RNA gene (rDNA). Ribosomal Database Project Classifier analysis revealed that among the 53 genera present, the Lactobacillus population drastically increased during fermentation, while the populations of other bacteria remained unchanged. Basic Local Alignment Search Tool analysis revealed that L. plantarum and L. brevis were the major species. Comparison with other fermented food microbiota indicated high product-dependency of the bacterial composition, which might have been due to the starter-free fermentation process.
Hygromycin B is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. Twenty-four hygromycin B-resistants mutants were isolated from sake yeast, and were divided into three different degrees of strength according to hygromycin B resistance. Three of four hygromycin B strongly resistant mutants produced increased amounts of isoamyl acetate in sake brewing test, although isoamyl alcohol levels remained unchanged. Many hygromycin B-resistants mutants showed higher E/A ratios than K-701 in culture with koji extract medium. Strain HMR-18 produced the largest amount of isoamyl acetate, and its alcohol acetyltransferase (AATFase) activity was 1.3-fold that of K-701. DNA microarray analysis showed that many genes overexpressed in HMR-18 were involved in stress responses (heat shock, low pH, and so on) but HMR-18 showed thermo- and acid-sensitivity. It was strongly resistant to hygromycin B and another aminoglycoside antibiotic, G418.
Continuous fermentation by retaining cells with a membrane-integrated fermentation reactor (MFR) system was found to reduce the amount of supplied sub-raw material. If the amount of sub-raw material can be reduced, continuous fermentation with the MFR system should become a more attractive process for industrialization, due to decreased material costs and loads during the refinement process. Our findings indicate that the production rate decreased when the amount of the sub-raw material was reduced in batch fermentation, but did not decrease during continuous fermentation with Sporolactobacillus laevolacticus. Moreover, continuous fermentation with a reduced amount of sub-raw material resulted in a productivity of 11.2 g/L/h over 800 h. In addition, the index of industrial process applicability used in the MFR system increased by 6.3-fold as compared with the conventional membrane-based fermentation reactor previously reported, suggesting a potential for the industrialization of this D-lactic acid continuous fermentation process.
A fungal strain, Penicillium sp. AZ, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12-carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that ammonia and nitrate nitrogen are available for PP-V biosynthesis, and that ammonia nitrogen was much more effective than nitrate nitrogen. Further, we isolated nitrate assimilation gene cluster, niaD, niiA, and crnA, and analyzed the expression of these genes. The expression levels of all these genes increased with sodium nitrate addition to the culture medium. The results obtained here strongly suggest that Penicillium sp. AZ produced PP-V using nitrate in the form of ammonium reduced from nitrate through a bioprocess assimilatory reaction.
A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1–33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently.
The utilization of 1-kestose (GF2) and nystose (GF3), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF2, whereas L. acidophilus and all the Bacteroides strains utilized both GF2 and GF3. Only the strains able to utilize both GF2 and GF3 had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF2 than for GF3, whereas those of the Bacteroides strains had higher activity for GF3 than for GF2. Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF3 medium was much higher than that of the cells grown in the GF2 medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF3 medium was almost the same as that of the cells grown in the GF2 medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.
Bacillus subtilis 168 is the only bacterium-based host serving for the cloning of giant DNA above 1.000 kbp. As rapid verification of the genome structure is crucial during the cloning process, six of 18-base sequence recognized by endonuclease I-SceI were sequentially created in the B. subtilis 168 genome. The established method and materials should be of use for other B. subtilis derivatives.
Agricultural waste water containing pesticides can reach the sea via rivers and estuaries, including brackish lakes. We studied the metabolic fate of methoxychlor [MXC; 1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] in a model system consisting of sediment and associated water collected from two sampling sites: a brackish lake and a freshwater river. MXC degraded rapidly and was finally mineralized in both sediment systems. The first step of degradation was dechlorination to yield 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane [de-Cl-MXC] or CN-replacement to yield 2,2-bis(4-methoxyphenyl)acetonitrile [MXC-CN], followed by O-demethylation. Although the metabolites were common to the two sediments, the dynamics of the metabolites over time were clearly distinct. In the brackish lake sediment, de-Cl-MXC accumulated transiently, whereas in the river sediment, it was rapidly converted to its demethylated metabolite. We also found that dechlorination and CN-replacement proceeded in autoclave-sterilized river sediment. In the river sediment, the abiotic reaction mediated by abundant humic acid and low oxygen level also appeared to contribute to the overall MXC metabolism.