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Heavy metals are harmful to human health. Therefore, we investigated the biosorption of heavy metals by lactic acid bacteria (LAB). Of all the tested heavy metals, biosorption by LAB was highest for mercury, followed by lead, cadmium, and finally arsenic. The viability of HCT-116 cells was reduced by half in the presence of 7.5 µg/mL mercury but recovered after the addition of selected LAB strains. HCT-116 cells showed increased superoxide dismutase and catalase activities, whereas glutathione peroxidase activities decreased significantly. Addition of Lactobacillus sakei TOKAI 57m recovered all antioxidant enzyme activities. Our results suggest that this strain can be used for cellular detoxification.

We investigated the content of phenolic compounds and antioxidant capacity of two batches of non-heated and heated leaves of the yacon cultivar “Andes no yuki”, grown in Japan. Lyophilized yacon leaves heated at 160°C for 20 min and 100°C for 60 min had a 1.96 to 9.69-times higher total phenolic content than that of the non-heated leaves. Heated leaves exhibited a 1.98 to 4.07-times higher antioxidant capacity than that of the non-heated leaves in three different free radical scavenging assays. Heated leaves were more efficient at attenuating the superoxide anion radical production in human granulocytic cells than the non-heated leaves. High-performance liquid chromatography analysis revealed that, in the heated leaves, the caffeic acid content was 2.13 to 3.64-times higher and the chlorogenic acid content was slightly lower than those in the non-heated leaves. Hence, heat processing may affect the active constituent contents in yacon leaves, potentiating its antioxidant capacity. Abbreviations: ABTS+: 2,2′-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; DPPH: 1,1-diphenyl-2-picrylhydrazyl, HPLC: high-performance liquid chromatography; NBT: nitroblue tetrazolium; O2−: superoxide anion; PMA: phorbol 12-myristate 13-acetate; PMS: phenazine methosulfate; TEAC: Trolox equivalent antioxidant capacity

Background Pharbitidis Semen (the seeds of Pharbitis nil), traditionally used as a purgative in Japan, China and Korea, contains a resin glycoside fraction named pharbitin, which is known as a purgative ingredient. Due to the complex nature of pharbitin, little is known about either the action on intestinal tension caused by resin glycoside itself or by its components. Methods In this study, we investigated the effects of pharbitin, the glycosidic acid fraction (pharbitic acid) and the aglycone fraction (phar-genin) generated from pharbitin on peristalsis of colon and ileum isolated from mice with the Magnus method. Results We demonstrated that pharbitin (3–30 μg/mL) concentration-dependently increased tonus of mice colon via acetylcholine receptors, its components phar-genin (1.27–12.7 μg/mL) and pharbitic acid (10–1000 μg/mL) also had the increment on colon tonus. On the other hand, ileum tension decreased in the presence of pharbitin. Conclusions The effects of resin glycoside of Pharbitidis Semen on colon tonus are different with those on ileum tonus isolated from mice. In the next step it is necessary to investigate details of its pharmacological mechanism.

The symptoms of foodborne histamine poisoning are similar to those of IgE-mediated food allergies. In this study, we investigated the histamine-binding capacity of lactic acid bacteria (LAB) strains as potential preventive agents against histamine poisoning. Histamine biosorption capacity was determined for 16 LAB strains. Leuconostoc mesenteroides TOKAI 51 m, Lactobacillus paracasei TOKAI 65 m, Lactobacillus plantarum TOKAI 111 m and Pediococcus pentosaceus TOKAI 759 m showed especially high biosorption rates and reached saturation within 30 min. Adsorption isotherms showed better conformance to the Freundlich model than to the Langmuir model. Analyses after heat, periodic acid and guanidine hydrochloride treatments suggested that histamine was bound to the bacterial cell surface. HPLC analysis revealed that exopolysaccharides produced by Lact. paracasei TOKAI 65 m strongly bound to histamine. In the detachment test with 1 mol l−1 HCl solution, the dissociation rate of histamine for Lact. paracasei TOKAI 65 m was

The heat susceptibility of starch granule structure has been considering as significant limitation of germinated black rice (GBR) using in food processing industry. Therefore, this study aimed to improve the physicochemical and antioxidation property as well as its effect on the probiotics of GBR by heat moisture treatment (HMT). Black rice germinated at 37.5 °C for 12, 24, and 36 h were studied. Ultrastructural image of each sample was visualized through scanning electron microscope. The results illustrated 24 h-GBR retain its former shape with rough surface. Subsequently, 24 h-GBR was structurally modified by HMT with moisture levels of 20% and 25% for 1 and 2 h. The results showed that pasting properties of HMT-treated GBR were improved. This was particularly on, GBR using HMT condition of 25% moisture for 2 h decrease in breakdown viscosity was shown. Moreover, phenolic content of HMT-treated GBR was higher than those of GBR. Besides, the number of the Lactobacillus paracasei TOKAI 13 was increased in GBR and HMT-treated GBR with counts of 10.08 ± 0.83 Log CFU/ml and 9.31 ± 0.33 Log CFU/ml, respectively, with significant increases in antioxidant property. Therefore, the HMT-GBR could be utilized as an alternative functional ingredient in food processing products.

Indoxyl, a derivative of indole originating from tryptophan, may undergo phase-II sulfate-conjugation pathway, thereby forming indoxyl sulfate (IS) in vivo. We previously reported that IS, a well-known uremic toxin, can increase the intracellular oxidation level and decrease the phagocytic activity in a differentiated HL-60 human macrophage cell model. Using the same cell model, the current study aimed to investigate whether indole and indoxyl (the metabolic precursors of indoxyl and IS, respectively) may cause macrophage immune dysfunction. Results obtained indicated that intracellular oxidation level and cytotoxicity markedly increased upon treatment with indole and indoxyl, in comparison with IS. Incubation of the cells with indole and indoxyl also resulted in attenuated phagocytic activity. Human serum albumin (HSA)-binding assay confirmed that tryptophan and IS, but not indole and indoxyl, could selectively bind to the site II in HSA. Collectively, the results indicated that indole and indoxyl may strongly down-regulate the phagocytic immune function of macrophages, whereas IS, formed upon sulfate conjugation of indoxyl, may exhibit enhanced HSA-binding capability, thereby reducing the adverse effects of indoxyl.

Small RNAs (sRNA) are non-cording RNAs composed of 50∼400 nt responsible for coordinating the adaption of Escherichia coli and other bacteria to changing environmental conditions, including pH and temperature. However, the role of sRNAs in lactic acid bacteria (LAB) has not yet been clarified. In this study, we used the Lactobacillus casei-Pediococcus group to evaluate the function of sRNAs in LAB, using RNA sequencing in the exponential growth phase and stationary phase to map and analyze sRNA fragments, which were categorized as Pediococcus pentosaceus and Lactobacillus paracasei. We evaluated the role of sRNAs in nutrient synthesis for cell growth in exponential growth phase and in protein and biofilm biosynthesis for cell body durability. During exponential growth, the sRNA fragments were found to be involved in the stress response in Pediococcus pentosaceus and in environmental adaption in Lactobacillus paracasei. The results suggest that the function of sRNA can be characterized from sRNA fragments using RNA sequencing during the exponential growth and stationary phases in Lactobacillus casei-Pediococcus group.

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine

We investigated the content of phenolic compounds and antioxidant capacity of two batches of non-heated and heated leaves of the yacon cultivar "Andes no yuki", grown in Japan. Lyophilized yacon leaves heated at 160°C for 20 min and 100°C for 60 min had a 1.96 to 9.69-times higher total phenolic content than that of the non-heated leaves. Heated leaves exhibited a 1.98 to 4.07-times higher antioxidant capacity than that of the non-heated leaves in three different free radical scavenging assays. Heated leaves were more efficient at attenuating the superoxide anion radical production in human granulocytic cells than the non-heated leaves. High-performance liquid chromatography analysis revealed that, in the heated leaves, the caffeic acid content was 2.13 to 3.64-times higher and the chlorogenic acid content was slightly lower than those in the non-heated leaves. Hence, heat processing may affect the active constituent contents in yacon leaves, potentiating its antioxidant capacity.

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp−) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.

The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9–8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins.

The gradual accumulation of heavy metals can have detrimental effects on health. Lactic acid bacteria (LAB) are common microbes used as probiotics; various LAB strains are consumed in food products, especially in fermented foods. Many studies have suggested that LAB with high affinity to harmful heavy metals can be used as efficient detoxification tools. Accordingly, it is important to test the biosorption of various heavy metals, e.g., cadmium, lead, arsenic, and mercury, by LAB. Here, I describe protocols to quantify the binding ability of LAB and to identify their heavy metal binding proteins.

The gradual accumulation of heavy metals can have detrimental effects on health. Lactic acid bacteria (LAB) are common microbes used as probiotics; various LAB strains are consumed in food products, especially in fermented foods. Many studies have suggested that LAB with high affinity to harmful heavy metals can be used as efficient detoxification tools. Accordingly, it is important to test the biosorption of various heavy metals, e.g., cadmium, lead, arsenic, and mercury, by LAB. Here, I describe protocols to quantify the binding ability of LAB and to identify their heavy metal binding proteins.

This study aimed to investigate the unique antioxidative effects of Japanese moringa products, herbal leaf tea and stem tea, using established free radical assays, focusing on superoxide anion (O2−) radical generation systems. Hot-water extracts from moringa teas resulted in different but lower scavenging activities than Trolox in four synthetic free radical models. Interestingly, these extracts further showed higher O2− radical scavenging effects than Trolox in the phenazine methosulfate-NADH-nitroblue tetrazolium and xanthine oxidase assay systems. Incubating human neutrophils in the presence of these tea extracts rather than Trolox effectively suppressed cellular O2− radical generation. Among the eight known phenolic constituents of moringa leaves, caffeic acid and chlorogenic acid may be responsible for the O2–specific radical scavenging capacity stronger than that of Trolox. These results suggest that moringa herbal teas are a good source of natural antioxidants for preventing O2− radical-mediated disorders. Abbreviations: O2−: superoxide anion; ROS: reactive oxygen species; H2O2: hydrogen peroxide; XOD: xanthine oxidase; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ABTS+: 2,2′-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; CPZ+: chlorpromazine cation; PMS: phenazine methosulfate; NBT: nitroblue tetrazolium; PMA: phorbol 12-myristate 13-acetate

Reactive oxygen species (ROS), such as hydroxyl and superoxide anion radicals, are highly reactive molecules derived from the metabolism of oxygen. ROS play positive roles in cell physiology, but they may also damage cell membranes and DNA, inducing oxidation that causes membrane lipid peroxidation and decreases membrane fluidity. Soymilk yogurt, which is soymilk fermented using lactic acid bacteria (LAB), is an excellent food item with numerous functional substances with antioxidant effects. In this study, the antioxidative activities of soymilk yogurt were investigated. Sixteen of the 26 tested LAB strains solidified soymilk. In antioxidant capacity tests for bacterial cells, Leuconostoc mesenteroides MYU 60 and Pediococcus pentosaceus MYU 759 showed the highest values in the oxygen radical antioxidant capacity (ORAC) and hydroxyl radical antioxidant capacity (HORAC) tests, respectively. The supernatant of soymilk yogurt made with Lactobacillus gasseri MYU 1 showed the highest ORAC and HORAC values. L. mesenteroides MYU 60, Lactobacillus plantarum MYU 74, Lactobacillus reuteri MYU 220, and P. pentosaceus MYU 759 showed significantly high N-acetylcysteine equivalent values compared with the control in a total ROS reducing assay (p

Many radionuclides were scattered by the explosion at the Fukushima Daiichi Nuclear Power Station. We examined whether lactic acid bacteria (LAB) can sorb cesium ions (Cs+) and strontium ions (Sr2+) for radioprotection. Many strains showed biosorption to Cs+ and Sr2+ using an in vitro mass-screening although each strain showed different sorption. We selected MYU 111, MYU 758, and MYU 759 strains that showed especially high biosorption to Cs+ and/or Sr2+. MYU 111 was identified as Lactobacillus plantarum, and MYU 758 and 759 were Pediococcus pentosaceus. The selected strains tended to show higher biosorption when using the buffer method compared to the culture method. Further, they showed high biosorption at a low concentration of 1 ppb Cs+ and Sr2+ (max 28.8% and 97.7% sorption, respectively). This is the first study where lactic acid bacteria are shown to have biosorption of Cs+ and Sr2+.

We developed an automated direct blotting system that completes the entire process of electrophoresis and electroblotting within 1–2 h. This system has high transfer reproducibility compared with the conventional semi-dry method. We also demonstrated the primary application of direct blotting of two-dimensional separated proteins, providing the expansion of protein spots.

Heavy metals cause various health hazards. Using lactic acid bacteria (LAB), we tested the biosorption of heavy metals e.g. cadmium (Cd) (II), lead (Pb) (II), arsenic (As) (III), and mercury (Hg) (II). Cd (II) sorption was tested in 103 strains using atomic absorption spectrophotometery (AAS). Weissella viridescens MYU 205 (1 × 10(8) cells/ml) decreased Cd (II) levels in citrate buffer (pH 6.0) from one ppm to 0.459 ± 0.016 ppm, corresponding to 10.46 μg of Cd (II). After screening, 11 LAB strains were tested using various pH (pH 4.0, 5.0, 6.0, 7.0) showing the sorption was acid sensitive; and was cell concentration dependent, where the Cd (II) concentration decreased from one ppm to 0.042 (max)/0.255 (min) ppm at 1 × 10(10) cells/ml. Additionally, the biosorption of Pb (II), As (III), and Hg (II) were tested using an inductively coupled plasma mass spectrometer (ICP-MS). The Hg (II) concentration was reduced the most followed by Pb (II) and As (III). Many of the bacterial cell surface proteins of W. viridescens MYU 205 showed binding to Hg (II) using the Hg (II) column assay. Having a CXXC motif, a ∼14 kDa protein may be one of the Hg (II) binding proteins. LAB biosorption may aid the detoxification of people exposed to heavy metals.

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 (p0.01). 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 (p0.01). 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.

Adhesion tests are complex, time-consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.

Aims: To identify and characterize a new adhesin-like protein of probiotics that show specific adhesion to human blood group A and B antigens. Methods and Results: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l−1 GHCl fraction extracted from Lactobacillus mucosae ME-340 contained a 29-kDa band (Lam29) using SDS–PAGE. The N-terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate-binding protein of the ATP-binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine-binding transporter. Conclusions: The adhesion of ME-340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin-like protein. Significance and Impact of the Study: Lactobacillus mucosae ME-340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.

The Kampo formula keishibukuryogan (KBG, Guizhifulingwan) is frequently used in traditional Japanese and Chinese medicine to treat several symptoms and manifests anti-inflammatory and scavenging effects. Nonalcoholic fatty liver disease (NAFLD) is a common manifestation of the metabolic syndrome and has the potential to evolve to liver cirrhosis through chronic inflammation and steatohepatisis (NASH). We have recently reported the KBG significant effectiveness on liver injury in a NASH animal model that prompted us to prescribe to KBG (TJ-25). We performed a retrospective study and reviewed the charts of outpatients who were prescribed KBG for 8-12 weeks due to non-liver-related symptoms (n= 11) over the past year to evaluate the clinical outcome. In six of these cases, biochemical and ultrasound signs of NAFLD were observed. KBG led to a significant reduction in liver injury tests and blood cholesterol but had no effects on body weight in all NAFLD cases. Further, liver tests and lipid profiles returned to baseline values when KBG treatment was stopped. On the basis of data on a small number of subjects, we suggest that the use of KBG is a safe complementary treatment in patients with NAFLD. While it is unlikely that Kampo formulas may substitute the current nutritional approaches to the metabolic syndrome, future studies should address the possibility of an additive effect, possibly through anti-inflammatory mechanisms.

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.

Total syntheses of sterically locked phycocyanobilin derivatives with a 15Z-anti or a 15E-anti CD-ring component were performed toward elucidation of the stereochemistry and function of the chromophore in phytochromes. In the course of the construction of a sterically locked 15E-anti CD-ring component employing 5-tosylpyrrolin-2-one derivatives as the D-ring, the Ts group was found to be rearranged under acidic conditions to give a mixture of regioisomers, both of which could be transformed into the same CD-ring precursor via detosylation with a base followed by Wittig-like coupling reaction. In addition, a sterically locked 15E-anti biliverdin derivative was also synthesized. Total syntheses of sterically locked phycocyanobilin derivatives bearing a 15Z-anti or a 15E-anti CD-ring component were performed towards elucidation of the stereochemistry and function of the chromophore in phytochromes.

The natural chromophore of most bacterial and fungal phytochromes is biliverdin (BV), which is incorporated in a covalent manner into the protein. Upon photoconversion between the red light-absorbing form Pr and the far-red light-absorbing form Pfr, the stereochemistry of the chromophore around the C 15 methine bridge changes from Z anti to E anti. Recombinant phytochromes Agpl and Agp2 from Agrobacterium tumefaciens were assembled with a set of synthetic chromophores, including 2,18-Et-BV, 3,18-Et-BV, and the doubly locked 5Ea15Ea-BV, 5Es15Ea-BV, 5Za15Ea-BV, and 5Zs15Ea-BV. In all chromophores, covalent bond formation is restricted. As shown by spectral changes and desalting column separation, all chromophores are bound to Agpl and Agp2. Adducts with 2,18-Et-BV and 3,18-Et-BV undergo normal photoconversion between Pr and Pfr. As opposed to typical phytochromes, the BV-Agp2 adduct converts from Pr to Pfr in darkness. However, the 2,18-Et-BV-Agp2 and 3,18-Et-BV-Agp2 adducts can undergo dark conversion from Pr to Pfr and Pfr to Pr, showing that ring A of the chromophore has a direct impact on the direction of dark conversion. The doubly locked chromophores were designed to probe for the stereochemistry of the C5 methine bridge in the Pfr form. The adducts with 5Es15Ea-BV and 5Zs15Ea-BV absorbed in the blue spectral range only. Therefore, the C5 E syn and Z syn stereochemistries are unlikely for the Pfr chromophore of Agpl and Agp2. According to our spectra, the Agp2 chromophore most likely adopts an E anti stereochemistry at its C5 methine bridge. Thus, during Pr to Pfr conversion, the C5 methine bridge of the chromophore might undergo a Hula-twist isomerization. In Agpl, the Pfr chromophore is most likely in the C5 Z anti stereochemistry. We propose that the stereochemistry of the C5 methine bridge might differ between different phytochromes, most particularly in the Pfr form.

Lactobacillus plantarum LA 318 is a potential probiotic strain isolated from normal human intestinal tissue that shows high adhesion to human colonic mucin mediated by the bacterial cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report the adhesion mechanism of the lactobacilli is in part due to GAPDH binding to human ABO-type blood group antigens expressed on human colonic mucin (HCM). After periodate oxidation of HCM, adhesion of L. plantarum LA 318 bacterial cells significantly decreased compared to normal HCM. A BIACORE binding assay of GAPDH to blood group antigens was then performed. High binding was observed to A and B group antigens, while binding to H group antigen was lower (P0.01). No interaction was observed between GAPDH and various monosaccharides. Furthermore, GAPDH binding to the B-trisaccharide biotinyl polymer (BP)-probe [Galalpha1-3 (Fucalpha1-2) Gal-] was significantly higher as compared to B-disaccharide, Lewis D-trisaccharide, 3-fucosyl-N-acetylglucosamine and alpha-N-acetylneuraminic acid BP-probes. The data suggests the trisaccharide structure is important in binding to the blood group antigens. The binding of GAPDH to HCM significantly decreased after incubation with NAD+. This suggests that the NAD binding domain on GAPDH may be related to binding to HCM.

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

Aims: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. Methods and Results: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115AE30 ± 12AE37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P

Because heavy metals cause various health hazards, we studied biosorption by Weissella viridescens MYU 205. MYU 205 showed high biosorption for Cd (II) and Hg (II) and was low for Zn (II). The Hg (II) biosorption rate was high at about 80%. Different biosorptions were shown for each metal after successive incubation. About 20% of the Zn (II) biosorption was observed after 3 h. Cd (II) biosorption increased in a time-dependent manner until 3 h, then gradually decreased. Hg (II) was immediately sorbed at 79.6 ± 4.7% and decreased at 3 h to 52.9 ± 2.6%, and then gradually increased to 77.8 ± 3.6%. Using heat-killed cells, the rate of biosorption of Zn (II) and Cd (II) decreased whereas Hg (II) tended to increase. The metal resistance was high, that is Zn (II) > Cd (II) > Hg (II); while the affinity was opposite where MYU 205 showed high affinity to Hg (II) and low affinity to Zn (II). Our data shows lactic acid bacteria may be powerful heavy metal sorbents for detoxification.

Background The “Liver damage” classification proposed by the Liver Cancer Study Group of Japan and Child-Pugh classification are both useful classifications for hepatic function. However, the factors responsible for the difference between the two classifications have not been fully investigated. Methods The medical records of 594 admissions of 220 patients with hepatocellular carcinoma (HCC) were retrospectively analyzed for encephalopathy, ascites, serum bilirubin and albumin, plasma retention rate (%) at 15 min after injection of 0.5 mg/kg of indocyanine green (ICG R15), and prothrombin time. Results Of 594 admissions, ICG R15 was tested in 337 (56.7%). The Child-Pugh classification was evaluated in all 594 admissions, but the “Liver damage” could be evaluated in 510 (85.9%) due to the lack of ICG R15 results. Of the 594 admissions, 372 (62.6%), 162 (27.3%), and 60 (10.1%) were Child-Pugh grade A, B, and C, respectively. Of the 510 admissions, 219 (42.9%), 202 (39.6%), and 89 (17.5%) were “Liver damage” grade A, B, and C, respectively. The grade of “Liver damage” was similar to that of Child-Pugh classification in 369 (72.4%), under-evaluated in 138 (27.1%), and over-evaluated in 3 (0.6%). The Child-Pugh classification was statistically a better classification for predicting outcome than “Liver damage”, but the “Liver damage” had better stratification ability than Child-Pugh classification in patients with relatively good liver function. Conclusions Although the “Liver damage” could not be evaluated in some cases due to the lack of ICG R15 results, this classification system is useful in the evaluation and prediction of outcome of patients with early-stage liver diseases.

The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2′-deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip.

The reaction of peptides containing an N-Boc- or N-Z-α-tosylglycine residue at the N-terminus with a variety of excess nitro compounds under basic conditions to give the nitro compound-adducts in good yields, followed by the elimination of nitrous acid from the adducts to afford the corresponding dipeptide containing α,β-didehydroamino acids with (Z)-configuration predominantly. Treatment of the same starting materials with aldehydes in the presence of base and tributylphosphine also gave a dipeptide containing α,β-didehydroamino acids with high (Z)-selectivity in satisfactory yields. The present method can be successfully applied to the synthesis of a protected leucine enkephalin analog.

Although anti-mitochondrial antibody (AMA) is the characteristic serological feature of primary biliary cirrhosis (PBC), its pathogenetic role remains unclear. We tested sera from 72 Japanese patients with histologically confirmed PBC for AMA by indirect immunofluorescence, anti-pyruvate dehydrogenase complex (PDC) by enzyme inhibition assay, immunoglobulin (Ig) G class anti-PDC by ELISA, and IgG, IgM, and IgA class anti-2-oxo-acid dehydrogenase complex (2-OADC) by immunoblotting. Of the 72 sera, 60 (83%), 50 (69%), 42 (58%), and 71 (99%) were positive for AMA by immunofluorescence, enzyme inhibition assay, ELISA, and immunoblotting, respectively. There was no significant correlation between histological stages and AMA by immunofluorescence, PDC inhibitory antibodies by enzyme inhibition assay, IgG class anti-PDC antibodies by ELISA, or IgG and IgM class anti-2-OADC by immunoblotting. IgA class anti-2-OADC by immunoblotting was more frequent in stages 2–4 than in stage 1 (P = 0.0083). Of the IgA class anti-2-OADC, anti-PDC-E2 (74 kDa) and anti-E3BP (52 kDa) were more frequent in stages 2–4 than in stage 1 (P = 0.0253 and 0.0042, respectively). Further examination of histopathological findings in 53 of 72 liver biopsy specimens showed that IgA class anti-PDC-E2 and IgA class anti-E3BP were associated with bile duct loss, and IgA class anti-PDC-E2 was also associated with interface hepatitis and atypical ductular proliferation. IgA is known to be secreted into the bile through biliary epithelial cells, implying that IgA class anti-PDC-E2 and E3BP may have a specific pathogenetic role during their transport into the bile by binding to their target antigen(s) in biliary epithelial cells, and this may be followed by dysfunction and finally destruction of biliary epithelial cells. Our present results suggest that these autoantibodies against 2-OADC detected by immunoblotting may be associated with the pathogenesis and pathologic progression of PBC.

Phytochromes are an important class of chromoproteins that regulate many cellular and developmental responses to light in plants. The model plant species Arabidopsis thaliana possesses five phytochromes, which mediate distinct and overlapping responses to light. Photobiological analyses have established that, under continuous irradiation, phytochrome A is primarily responsible for plant's sensitivity to far-red light, whereas the other phytochromes respond mainly to red light. The present study reports that the far-red light sensitivity of phytochrome A depends on the structure of the linear tetrapyrrole (bilin) prosthetic group. By reconstitution of holophytochrome in vivo through feeding various synthetic bilins to chromophore-deficient mutants of Arabidopsis , the requirement for a double bond on the bilin D-ring for rescuing phytochrome A function has been established. In contrast, we show that phytochrome B function can be rescued with various bilin analogs with saturated D-ring substituents.

The serum reaction to anti-2-oxo-acid dehydrogenase complex (2-OADC) enzymes, the antigens recognized by antimitochondrial antibodies (AMA), can be detected by immunoblotting in patients with liver diseases other than primary biliary cirrhosis (PBC), who are negative for AMA by conventional indirect immunofluorescence. Whether the presence of anti-2-OADC is related to PBC or represents preclinical PBC in such patients is obscure at present. We examined the immunoreactivity of AMA by immunofluorescense, immunoblotting, and enzyme inhibition assay in serum samples from 59 patients with liver diseases other than PBC and 71 healthy subjects. We also examined the clinical course of the patients in whom a positive result was obtained to elucidate whether such reaction was a "true" or "false" phenomenon. None of the 130 sera was positive for AMA by indirect immunofluorescence or for anti-pyruvate dehydrogenase complex (PDC) by enzyme inhibition assay. However, seven of 71 (10%) sera from healthy subjects contained weak IgG class antibody to PDC-E2 (four sera) or E2 subunit of branched-chain oxo-acid dehydrogenase complex (BCOADC-E2) (three sera). Of the 59 sera from patients with liver diseases other than PBC, four (7%) reacted against 2-OADC by immunoblotting. Of these, three sera were from patients with chronic hepatitis C virus (HCV) infection, and contained IgG class autoantibody to BCOADC-E2. The serum reactivity to BCOADC-E2 detected by immunoblotting in these three patients diminished after absorption with recombinant BCOADC-E2 fusion protein. During the 3-5 year follow-up period, AMA by immunofluorescence and anti-PDC activity by enzyme inhibition assay were always negative in these three patients. The other one serum was from patient with alcoholic cirrhosis, and contained IgM class autoantibody to E3 binding protein (E3-BP). This patient did not develop PBC during the following 2 years. Our results showed that anti-2-OADC antibodies could be detected in some patients with liver diseases other than PBC, and even in healthy individuals. The clinical significance of the presence of these serum reactions is obscure at this stage, but the production of anti-BCOADC-E2 may be linked to the presence of HCV in certain patients. Further prospective studies of larger population should clarify whether anti-2-OADC reaction can precede the clinical development of PBC.

Ethyl N-Boc- and N-Z-α-tosylglycinates were reacted with a variety of aldehydes in the presence of tributylphosphine and a base to afford the corresponding α,β-didehydroamino acid derivatives with high (Z)-selectivity in good yields. Moreover, ethyl (4S)- and (4R)-2-(N-Boc-amino)-4,5-isopropylidenedioxy-2-pentenoates prepared by the present method were converted to (2S, 4S)- and (2R, 4R)-4-hydroxyprolines, respectively.

Two-dimensional gel electrophoresis (2D-PAGE) has been a commonly used technique for protein expression studies. Application of a fluorescent dye in 2D-PAGE, namely 2D-DIGE, has improved the performance of 2D-PAGE in terms of sensitivity, reproducibility and throughput. However, 2D-PAGE still requires a degree of skill, and is considerably time-consuming. Recently, a novel automated 2D-PAGE machine, the Auto2D, was developed for use in conventional protein expression studies. Here, we examined the performance of the Auto2D for 2D-DIGE from the viewpoint of proteome coverage, reproducibility, and throughput. We found that a single 2D image of osteosarcoma cells contained 521 protein spots. By running an identical sample, we found that the system reproducibility was high and the intensity of more than 409 protein spots was scattered within a 2-fold range of differences between experiments, with a correlation coefficient of at least 0.60. We also found that a single 2D-PAGE run took 140 min, and that 3 gels could be run per day. We conclude that the Auto2D can be used for conventional protein expression studies. On the other hand, as the number of observed proteins was quite limited, and only three gels can be run per day per device, the Auto2D may not be suitable for a large-scale proteomic study.

Despite rapid progress in methods for analyzing radiation effects, much remains to be learned about the mechanisms and processes of radiation-induced immunological dysfunction. Among 17,899 sera obtained from atomic bomb survivors in Nagasaki, Japan, sera from 484 participants who complied with a reexamination for alkaline phosphatase (ALP) were tested for antimitochondrial antibody (AMA) by indirect immunofluorescence, and autoantibodies against 2-oxo-acid dehydrogenase complex (2-OADC) by immunoblotting to investigate the prevalence of primary biliary cirrhosis (PBC). Of these 484 sera, 28 (5.8%) were seropositive for AMA. The 484 participants were divided into three groups according to distance from the hypocenter: 72 who were exposed within 1999 m (closest group), 368 from 2000 to 5999 m (intermediate distant group), and 44 outside 6000 m (distant group). The positivity rates for AMA in these three groups were 6/72 (8.3%), 22/368 (6.0%), and 0/44 (0%), respectively (P =.08). Furthermore, high titers ( > 1:320) of AMA were observed in 3/6 (50%) AMA-positive sera from the closest group, in contrast to 4/22 (18%) from the intermediate distant group, although there was no significant correlation between AMA titer and distance from the hypocenter (P =.07). Of these 28 AMA-positive sera, 11 (39%) were from participants who had already been diagnosed with PBC, and 25 (89%) contained antibodies against at least one component of 2-OADC enzymes by immunoblotting. Therefore, the prevalence of PBC was estimated to be at least 615 cases per million (792 per million women). Our results suggest that the prevalence of PBC in atomic bomb survivors in Nagasaki is higher than that reported for the general population in Japan, and a further survey of the environmental factors, including radiation exposure, that predispose to PBC would be needed for understanding this disease of unknown etiology.