The enzymes from hyperthermophiles are generally extremely thermostable and lose little or no activity during long periods under a variety conditions. This high stability is very attractive, in that it gives the enzymes potential for use in numerous bioprocesses. My research group has investigated this high stability from the viewpoint of the relationship between function and structure. In this review, I describe the molecular mechanism underlying the extreme stability of unboiled NAD-dependent glutamate dehydrogenase from the hyperthermophile Pyrobaculum islandicum. I also describe the activation of the inactive recombinant enzyme produced in mesophilic Escherichia coli from the viewpoint of the relationship between structure and activity.

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Kimchi is a well-known traditional Korean food. Its geographical origins can be determined by its biochemical composition. This study identified the biochemical compositions of kimchi extracts from Korea and China by ¹H nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analysis. Principal component analyses (PCA) clearly discriminated between extracts prepared in the two countries. The identified metabolites, including amino acids, organic acids, sugars, and ethanol, contributed to discriminating the geographical origin of kimchi extracts. Furthermore, differences in composition by origin were predicted with high accuracy in external validation models. These results establish biochemical profiles for kimchi extracts, and indicate that metabolomics can be used in the discrimination of food origins.

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The antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, were successfully detected in the aorta of spontaneously hypertensive rats after orally administering these peptides by a guanidine-thiocyanate treatment to prevent proteolysis. Cy3-labeled versions of both peptides were localized in the endothelial cells of arterial vessels in the rats. The accumulation of both peptides in the endothelial cells suggested in vivo inhibitory activity of the angiotensin I-converting enzyme.

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Optically pure (+)- and (−)-boronolides were stereoselectively synthesized from yeast reductive products which had been obtained by yeast reduction of one common racemic substrate. The lactone structure of boronolide was constructed by Baeyer-Villiger oxidation. The stereochemistry of the yeast reduction products was studied to obtain the stereocenters at 5 positions of the dodecanolides of (+)- and (−)-boronolides. The stereochemistry of the 6 and 7 positions was obtained by AD-mix-α or β oxidation. The chiral center at the 8 position was constructed by employing (R)-(+)- or (S)-(−)-2-methyl-CBS-oxazaborolidine reduction or the Mitsunobu reaction. The plant growth-inhibitory activity against Echinochloa crusgalli L. of naturally occurring (+)-boronolide was higher than that of the (−)-boronolide.

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Esterified ceramides, (2S,3R,4E)-2-[(30'-stearoyloxy)triacontanamido]octadec-4-ene-1,3-diol (2) and (2S,3R,4E,6R)-2-[(30'-stearoyloxy)triacontanamido]octadec-4-ene-1,3,6-triol (4), are minor components of the human stratum corneum. We synthesized these ceramides by employing olefin cross metathesis as the key reaction for constructing their ω-hydroxytriacontanoyl part (13).

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Four new meroterpenoids, named chrodrimanins D–G (4–7), and one known compound, renamed chrodrimanin H (8), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with the YO-2 strain of Talaromyces sp. Their structures were elucidated by spectroscopic methods. Chrodrimanins D (4), E (5), and F (6) showed insecticidal activity against silkworms with respective LD₅₀ values of 20, 10, and 50 µg/g of diet.

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The new anthraquinone derivative, 6,8-di-O-methylaverantin (1), together with six known congeners (2–7), were characterized from a culture extract of the marine algous fungus, Aspergillus versicolor EN-7. Their structures were elucidated by spectroscopic methods. A bioassay showed compounds 1, 2 and 5 to have weak inhibition against Escherichia coli, while compound 5 also displayed weak inhibition against Staphylococcus aureus.

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An efficient chemo-enzymatic procedure for the synthesis of (R)-3-hydroxypentanenitrile (1) with over 99% enantiomeric excess using two enzymatic reactions was successfully established. Initial enantioselective enzymatic reduction of 3-oxopentanenitrile with reductase S1 gave (R)-1 with an 81.5% ee which was then converted to (R)-1-(cyanomethyl) propyl n-butyrate (3b). Subsequent lipase-catalyzed enantioselective hydrolysis of 3b gave (R)-1 in a high yield with over 99% ee.

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Although Dmrt7 has been reported to be essential for male spermatogenesis, the molecular mechanism underlying pachytene spermatogenesis by Dmrt7 is not known. In the present study, by detailed analysis of Dmrt7 protein distribution in spermatocytes in the first wave of spermatogenesis, we clarified the profile of Dmrt7 expression and localization in pachytene spermatogenesis. Dmrt7-deficient spermatocytes were arrested in the pachytene stage, followed by apoptosis. We analyzed to determine whether every event in the spermatogenesis at the Dmrt7-deficient mice progressed normally, because in several gene knockout mice with spermatogenic arrest described in the previous reports impairments of these events often appeared. Mutant mice showed normal synapsis and XY body formation, while impairment of meiotic sex chromosome inactivation (MSCI), decreased expression of backup genes, and increased expression of retrotransposons indicated incomplete meiotic recombination.

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An α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a bi-functional enzyme showing both α2,3-sialyltransferase and α2,3-linkage specific sialidase activity. To date, the crystal structures of several sialyltransferases have been solved, but the roles of amino acid residues around the catalytic site have not been completely clarified. Hence we performed a mutational study using α2,3-sialyltransferase cloned from P. phosphoreum JT-ISH-467 as a model enzyme to study the role of the amino acid residues around the substrate-binding site. It was found that a mutation of the glutamic acid at position 342 in the sialyltransferase resulted in a loss of sialidase activity, although the mutant showed no decrease in sialyltransferase activity. Based on this result, it is strongly expected that the Glu342 of the enzyme is an important amino acid residue for sialidase activity.

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To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

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The signalling molecule 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one (DIF-1) is required for differentiation and pattern formation in Dictyostelium discoideum development. DIF-1 is synthesized by three enzymes, a hybrid polyketide synthase, a flavin-dependent halogenase, and a des-methyl-DIF-1 methyltransferase. The genome data on the related species D. purpureum are now public. Using this genome information, des-methyl-DIF-1 methyltransferase of D. purpureum was identified, and was named Dp dmtA. Overexpression of Dp dmtA complemented the defects in basal disc formation and lower cup formation in a dmtA knock-out mutant of D. discoideum. This indicates that Dp dmtA has the same function as D. discoideum dmtA and compensates for loss of the dmtA gene in the D. discoideum dmtA mutant. The materials released in the medium by D. purpureum contained stalk-inducing activity with the same retention time as that of DIF-1 in HPLC fractionation. This indicates that the stalk-inducing signal of DIF-1 and des-methyl-DIF-1 methyltransferase are conserved in D. purpureum.

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In HEK293 cells, exposure to various NAD(P)H oxidants, including phenazine methosulfate (PMS), that non-enzymatically oxidize intracellular NAD(P)H to NAD(P), decreased hypoxia-induced hypoxia-inducible factor 1 (HIF-1α) accumulation. RT-PCR and cycloheximide inhibition experiments indicated that PMS-induced HIF-1α decrease is involved in post-translational degradation during hypoxia. The decrease in HIF-1α caused by PMS was not eliminated by proteasome inhibitor MG132. Moreover, the increase in HIF-1α induced by exposure to MG132 alone in normoxia was diminished by PMS. In contrast, calpastatin peptide, a calpain inhibitor, fully prevented PMS-induced reduction in HIF-1α in hypoxic cells. These data suggest that the decreased stability of HIF-1α induced by PMS is due to the activation by PMS of a protein degradation system that is independent of the ubiquitin-proteasome pathway.

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We have reported the construction of 1 Mb reduced genome Escherichia coli MGF-01 by a 28-step operation. This time, transcriptome analysis of MGF-01 was performed. Although the transcriptome profiles of the exponential phase in parental strain W3110red were well-conserved in MGF-01, the rspAB operon was highly expressed. A LacZ reporter assay of a series of stepwise deletion strains prepared in the course of MGF-01 construction indicated that rspA was highly expressed after the 5th step. Further analysis indicated that Δ29, one of the deleted regions at the 5th step, relates to an increase in rspA expression, and that transcriptional regulator ydfH, in the Δ29 region, is responsible for the expression of rspA, gel shift assay indicated that YdfH bound directly to the upstream region of rspA. Based on these results, it was concluded that YdfH is a transcriptional repressor of the rspAB operon.

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A novel esterase showing activity specific for esters of aryl-carboxylic acids was discovered in Sporosarcina sp. nov., which was identified by the 16S rDNA sequencing method in addition to morphological and physiological analyses. The aryl-carboxylesterase (named EstAC) was purified 780-fold from crude cell extracts by a 5-step procedure. EstAC was characterized as a monomeric protein with a molecular weight of 43,000, an optimum pH of around 9.0, and an optimum temperature of 40 °C. The pH optimum and the effects of inhibitors together with an internal amino acid sequence suggested that EstAC is a member of family VIII esterases. EstAC was found to be highly active on a wide variety of substrates such as alkyl benzoates, alkyl phenylacetates, ethyl α- or β-substituted phenylpropionates, dialkyl terephthalates, dimethyl isophthalate, and ethylene glycol dibenzoate. However, monomethyl terephthalate was not hydrolyzed. It was suggested that EstAC had 4-hydroxybenzoyl and cinnamoyl esterase activities as well.

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This present study is the first attempt to report on the purification and characterization of a chitinase from the stomach of the red scorpionfish Scorpaena scrofa. A 50-kDa chitinase (SsChi50) was purified to homogeneity, and matrix assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) analysis showed that SsChi50 was a monomer with a molecular mass of 50,103 Da. The 25 N-terminal residues of SsChi50 displayed high homology with family-18 chitinases. Optimal activity was obtained at pH 5.0 at 80 °C. SsChi50 was stable at pH and temperature ranges of 3.0 to 7.0 and 70 to 90 °C for 48 and 4 h respectively. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg²⁺, and Hg⁺ completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-NP-(GlcNAc)_n (n=2 –4) was p-NP-(GlcNAc)₂ > p-NP-(GlcNAc)₄ > p-NP-(GlcNAc)₃. Our results suggest that the SsChi50 enzyme preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)_n. This enzyme obeyed Michaelis-Menten kinetics, the K_m and k_cat values being 0.412 mg, colloidal chitin mL⁻¹ and 5.33 s⁻¹ respectively. An in vivo bioinsecticidal assay was developed for SsChi50 against Callosobruchus maculatus adults. The enzyme showed bioinsecticidal activity toward Callosobruchus maculatus, indicating the possibility of using it in biotechnological strategies for insect management for stored cowpea seeds.

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Diabetic nephropathy is a major complication of diabetes and tubulointerstitial fibrosis is one of its manifestations. This study aimed to clarify the pathogenicity of advanced glycation endproducts (AGEs) toward NRK-52E, a tubular epithelial cell line. The AGE-exposed cells significantly increased gene expression of transforming growth factor beta, plasminogen activator inhibitor-1, and tissue transglutaminase, and a medium conditioned by them showed strong potential to recruit macrophages, partly through a chemokine, monocyte chemoattractant protein-1. Albumin denatured by maintenance at 37 °C for 120 d exhibited similar activities, but they were lower than those of the AGEs. Thus, AGEs generated in diabetic patients might exacerbate fibrosis in the kidneys directly through renal epithelial cell stimulation, and indirectly by recruitment of macrophages.

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We screened a novel sphingosine 1-phosphate receptor type 5 motif-containing gene, LOC290876, from maturing rat testes by differential display. Gene expression was testis-specific, increased at week 7, and continued for 15 weeks. PCR analysis clarified two gene transcript isoforms, which were expressed at the same level in all samples detected in Northern blot. The deduced amino acid sequences of the two isoforms revealed differences in carboxyl terminal sequences. Gene and protein expression in the testes was dominant in the spermatocytes, and protein expression was localized to the nucleus. Taken together, these findings suggest that the LOC290876-encoded gene product is not involved in sphingosine signaling, but has distinct roles in the nucleus during the processes of spermatocyte maturation and meiosis producing spermatids.

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The visualization of nuclear architecture, which changes dynamically depending on the physiological and the pathological situation, remains an important challenge. Here we report that exposure of fluoresceinisothiocyanate-labeled polyethylenimine (FITC-PEI) to digitonin-permeabilized cervical cancer HeLa cells enable rapid detection of the morphology of the nuclear rim, the nucleolus, and mitotic chromosomes. This simple detection strategy can aid in scientific investigation for both basic research and diagnostic purposes.

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Chitosan (CHT)-induced stomatal closure was inhibited by an MAPKK inhibitor, PD98059, and was impaired in mpk9 mpk12 but not in mpk9 or mpk12. CHT induced the production of reactive oxygen species, cytosolic alkalization, and cytosolic Ca²⁺ oscillation in mpk9 mpk12. These results suggest that MPK9 and MPK12 are involved in CHT-induced stomatal closure.

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We produced a single deuterated lachrymatory factor (propanthial S-oxide, m/z=91) in a model reaction system comprising purified alliinase, lachrymatory factor synthase (LFS), and (E)-(+)-S-(1-propenyl)-L-cysteine sulfoxide ((E)-PRENCSO) in D₂O. Onion LFS reacted with the degraded products of (E)-PRENCSO by alliinase, but not with those of (Z)-PRENCSO. These findings indicate that onion LFS is an (E)-1-propenylsulfenic acid isomerase.

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Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H⁺-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

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We examined the effects of nucleoside monophosphates on the dissociation of actomyosin into myosin and actin. GMP was effective only among GMP, CMP, dTMP, and UMP. Hence we concluded that purine-based nucleoside monophosphates such as GMP, AMP, and IMP are effective, incorporating this with our previous results (Okitani A et al., Biosci. Biotechnol. Biochem., 72, 2005–2011 (2008)). Then we examined whether IMP enhances the extraction of myosin and actin as well as pyrophosphate (KPP), using homogenates of pork with 9 volumes of 0.3, 0.4, and 0.5 M NaCl solutions containing 0–36 mM IMP or 0–9 mM KPP. Maximum extractability, about 70% for both proteins, was attained by means of NaCl solutions containing 36 mM IMP. These values were comparable to the maximum values, about 90% for myosin and 50% for actin, attained by means of solutions containing 9 mM KPP. Hence we concluded that IMP enhances the extraction of myosin and actin from porcine meat.

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A chemical investigation of the n-butanol fraction of the inner bark of Betula platyphylla led to the isolation of seven diarylhepanoids, (−)-centrolobol (1), aceroside VII (2), aceroside VIII (3), (3R)-1,7-bis-(4-hydroxyphenyl)-3-heptanol-3-O-[2,6-bis-O-(β-D-apiofuranosyl)-β-D-glucopyranoside (4), 1,7-bis-(4-hydroxyphenyl)-5-hepten-3-one (5), platyphyllone (6) and platyphylloside (7). The antifibrotic effects of these isolates were evaluated with HSC-T6 cells by assessing cell proliferation. Among them, compounds 1, 2, 5 and 6 significantly inhibited the proliferation of HSCs in a dose-dependent manner at concentrations from 10 µM to 100 µM. Compound 5 in particular dramatically decreased the collagen content and increased the Caspase-3/7 activity. Taken together, the antifibrotic activity of B. platyphylla and its constituents might suggest therapeutic potential against liver fibrosis.

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There are very limited data concerning the influence of low-level trans fatty acid (TFA) intake on blood lipid levels. In this study, correlation of total and diene TFA intake with serum cholesterol levels was studied in young Japanese women. The mean intakes of total and diene TFAs were 0.36% and 0.05% of energy, respectively. There was a significant correlation between total fat intake and TFA intake. TFA intake was significantly correlated with erythrocyte TFA content. Total TFA intake was not correlated with total, LDL- or HDL-cholesterol levels. No correlatuon was found between diene TFA intake and cholesterol level. Total and diene TFA intake were not correlated with hemoglobin A1c or C-reactive protein levels. These results suggest that the average TFA intake of young Japanese women does not adversely affect serum cholesterol levels.

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Elderberry (Sambucus nigra L.) has traditionally been used for treating influenza and colds. We evaluated the antiviral effect of concentrated juice of elderberry (CJ-E) on the human influenza A virus (IFV). CJ-E had a relatively strong effect on IFV-infected mice, although its anti-IFV activity was weak in a cell culture system. The in vivo anti-IFV activities of the fractions were determined after separating CJ-E by ultrafiltration and anion-exchange chromatography. Oral administration of the high-molecular-weight fractions of CJ-E to IFV-infected mice suppressed viral replication in the bronchoalveolar lavage fluids (BALFs), and increased the level of the IFV-specific neutralizing antibody in the serum, as well as the level of secretory IgA in BALFs and feces. Fr. II from high-molecular-weight fraction HM, which contained acidic polysaccharides, showed relatively strong defense against IFV infection. We conclude that CJ-E had a beneficial effect by the stimulating immune response and preventing viral infection.

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Such rice cultivars as Japonica, Japonica-Indica hybrid, Javanica and Indica, were evaluated for their main chemical components (amylose content and protein content), pasting property of rice flour (consistency), physical property of the cooked rice grains (adhesion, L3), and enzyme activities (cellulase and xylanase). The amylose content, cellulase activity and xylanase activity showed significant positive or negative correlation with the pasting property (consistency) of rice flour (r=0.89, r=0.58, r=0.70, respectively) and with the physical property of the cooked rice grains (adhesion, L3: r=−0.51, r=−0.61, r=−0.71, respectively) at the level of 1%. Endogenous xylanase and cellulase played important roles to determine the texture of the cooked rice grains similarly to the amylose content. Part of the DNA sequences of the α-glucosidase gene differed among the Japonica, Japonica-Indica hybrid and Indica subspecies. We found discriminative DNA bands appearing by PCR, corresponding to 1,4-β-xylanase and endo-1,4-β-glucanase 13 in the case of Indica rice, Indica-Japonica hybrid rice, and Javanica rice (non-Japonica subspecies). The equation for estimating the physical property (adhesion) of cooked rice grains by PCR was improved by adding novel primers related to the cell-wall-degrading enzymes.

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The antihypertensive effect of a single oral administration of a boysenberry seed polyphenol extract to spontaneously hypertensive rats was evaluated at different doses (100 and 200 mg/kg), and a significant decrease in systolic blood pressure (SBP) was observed up to 6 h post administration. The extract was separated into proanthocyanidin-rich and ellagitannin fractions by solvent partition. A significant decrease in SBP was observed only after administering the proanthocyanidin-rich fraction, and this decrease was abolished by an N^G-nitro-L-arginine methyl ester (L-NAME) injection. An analysis of the orally absorbable components showed that intact dimeric and trimeric procyanidins and propelargonidins were detectable in the plasma with a maximal concentration 2 h post administration. The vasorelaxant activity of the extract was also confirmed by in vitro assay using rat aorta rings. These results suggest that proanthocyanidins (PAs) in boysenberry seeds may have played an important role in the observed antihypertensive effect.

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We compared the colonization of lactobacilli in the stomachs of mice fed nonpurified and purified diets and examined to determine whether the expression of Toll-like receptor 2, which is involved in the recognition of lactobacilli, is influenced by diet. Female BALB/c mice were fed a nonpurified or a purified diet for 2 weeks. Conventional cultivation and cultivation-independent molecular biological analysis based on the 16S rRNA gene sequence revealed that the number of lactobacilli associated with the gastric tissue was significantly higher in the mice fed the nonpurified diet than in those fed the purified diet. Sequencing analysis indicated that L. gasseri and L. johnsonii were predominant Lactobacillus species associated with the gastric tissue of the mice fed the nonpurified diet. The mRNA levels of Toll-like receptor 2, but not of 9, in the gastric tissue were significantly higher in the mice fed the purified diet than in those fed the nonpurified diet. We propose that nonpurified and purified diets have different impacts on gastric microbiota, which can in turn influence the expression of Toll-like receptor 2 in the stomach.

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To determine whether ingestion of citronellal decreases the attractive power of the male mouse urinary odor, female mice were used in preference tests. A series of tests revealed that the female mice preferred voided urine odors from aged mice over those from younger adult mice. However, exogenous citronellal directly inhibited the advantage of the aged males with regard to attraction.

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Supplementation to an AIN93G-based diet of tocotrienol (T3) for 13 weeks administered to Fischer 344/slc rats showed a safety profile with no side effects. Dose-dependent T3 levels were detected in many tissues. Under the present experimental conditions, a continuous intake of the T3 concentrate would be safe in the rats as long as the T3 content was less than 0.20% of the dietary intake.

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A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

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Hydrogenobacter thermophilus is a chemolithoautotroph that utilizes not only hydrogen (H₂) but also thiosulfate as sole source of energy and assimilates carbon dioxide via the reductive tricarboxylic acid (RTCA) cycle. We systematically carried out transcriptome analysis of metabolic enzymes in both H₂- and thiosulfate-grown H. thermophilus cells. The analysis indicated that the expression of hydrogenase genes is repressed under thiosulfate oxidation conditions as compared with H₂ oxidation conditions. This was confirmed by enzyme assay. In contrast, some genes for sulfur metabolism, including sox genes, showed almost the same expression levels under both conditions. In addition, the genes for the RTCA cycle showed high expression levels under both conditions. It was suggested that sulfur metabolism and the RTCA cycle function as forms of basal metabolism, and H₂ oxidation is inducible. Switching of H₂ oxidation can be advantageous for the lifestyle of this bacterium in nature.

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The replication region of the 111-kb circular plasmid pKNR from Rhodococcus opacus B-4 was identified. A PCR-based deletion analysis using the λ Red recombination technique followed by restriction digestion and PCR-amplification analyses revealed that a 2.5-kb fragment covering one putative open reading frame (ORF) was involved in the replication of pKNR. The product of this ORF showed significant similarity to a functionally unknown protein encoded in the replication region of the 70-kb circular plasmid of Clavibacter michiganensis and to ones in other bacterial large circular plasmids. These observations suggest that the product of the identified ORF and its orthologs can serve as novel replication proteins for large circular bacterial plasmids.

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Forty-three fungal producers for glycolipid biosurfactants, mannosylerythritol lipids (MELs), were isolated from leaves and smuts of sugarcane plants. These isolates produced MELs with sugarcane juice as nutrient source. The strains were taxonomically categorized into the genera Pseudozyma and Ustilago on the basis of partial sequences of the ribosomal RNA gene.

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Glutamate is a major free amino acid in cyanobacteria, but its transport properties remain largely unknown. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica, contained a sodium dependent glutamate transporter (ApGltS). The deduced amino acid sequence of ApGltS exhibited low homology (18–19% identity) to GltS from Synechocystis sp. PCC 6803 (slr1145) and Escherichia coli. The predicted ApGltS consisted of 476 amino acid residues with a molecular weight of 50,976 Da. As analysed by hydropathy profiling, ApGltS contains 11 transmembrane segments. The ApgltS gene was isolated and expressed in E. coli ME9107, which is deficient in glutamate uptake. ME9107, expressing ApGltS, took up glutamate and its rates increased with increasing concentrations of NaCl. Kinetics studies revealed that ApGltS is a high-affinity glutamate transporter with a K_m of about 5 µM. The presence of 0.5 M NaCl in the assay medium increased V_max by about 3-fold. Competition experiments revealed that glutamate, glutamine, aspartate, and asparagine inhibited glutamate uptake. The level of mRNA for ApgltS was higher in A. halophytica grown at high salinity. Under high salinity conditions supplemented with glutamate, A. halophytica showed a significant increase in intracellular glycine betaine.

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Glycoglycerolipids were isolated from seaweed-associated marine actinomycete strain PK206-15, which was identified as Streptomyces coelescens based on 16S rDNA sequence analysis. The compounds were isolated by silica gel chromatography and high performance liquid chromatography. The structures were analyzed by nuclear magnetic resonance, high resolution-electrospray ionization-mass spectrometry, and methanolysis. The compounds were identified as 2R-1,2-di-12-methylhexadecanoic acid-3-O-[β-D-galactopyranosyl-(1''-6')-O-β-D-galactopyranosyl]-glycerol, 2R-1-12-methylhexadeca noic acid-2-hydroxyl-3-O-[β-D-galactopyranosyl-(1''-6')-O-β-D-galactopyranosyl]-gly cerol, 2R-1,2-di-12-methylhexadecanoic acid-3-O-β-D-galactopyranosyl-glycerol, and 2R-1,2-di-14-methylhexadecanoic acid-3-O-β-D-galactopyranosyl-glycerol. These were active against the following fouling organisms: zoospores of Ulva pertusa, the diatom Navicula annexa, the mussel Mytilus edulis, and fouling bacteria with an EC₅₀ range of 0.005–0.2 µg/mL.

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Practical removal of radioactivity from polluted soil in Fukushima, Japan was done using a photosynthetic bacterium, Rhodobacter sphaeroides SSI, immobilized in alginate beads. The beads were put in a mesh bag and soaked in which soil was suspended (5 kg of soil/10 L of tap water). The radioactivity of the broth decreased by 31% after 15 d of aerobic treatment. When lactic acid bacterial culture broth was added to the suspend broth, about 50% of the radioactivity was transferred to a suspend broth fraction consisting of small particles from the soil after 3 d of fermentation and 20 s of sedimentation. The results suggest that organic matter in the soil was decomposed by anaerobic digestion and lactic acid fermentation simultaneously, and was then transferred into the liquid as small particles. With combined treatment by anaerobic digestion and lactic acid fermentation for 5 d and immobilized bead aerobic treatment for an additional 19 d, the radioactivity of suspend broth decreased by 66%. The radioactivity of the original soil (10.56 µSv/h) ultimately decreased by 67% (3.52 µSv/h) after the combined treatment.

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