Novel Ree1 regulates the expression of ENO1 via the Snf1 complex pathway in Saccharomyces cerevisiae.

Using cDNA microarray analysis, we found that the mRNA of YJL217W and several other genes related to cell wall organization and biogenesis were up-regulated by galactose in Saccharomyces cerevisiae early during the induction process. YJL217W is also known as REE1 (Regulation of Enolase I). Both the Gal4 regulatory region and the Mac1 binding domain were found on the upstream region of REE1, and the expression of REE1 was up-regulated by galactose but not by glucose. The up-regulation of REE1 by galactose was not observed in the Deltagal4 strain. From the two-hybrid analysis, we found that Ree1 physically interacted with Gal83. Furthermore, from 2-D gel electrophoresis we found that the deletion of REE1 resulted in the up-regulation of Eno1. From Western blotting, we learned that the expression of Eno1 in the Deltaree1 strain was different from that in wild-type strains and that Eno1 expression was not changed by glucose stimulation. Taken together, these results suggest that Ree1p functions in the galactose metabolic pathway via the Gal83 protein and that it may control the level of Eno1p, which is also affected by the Snf1 complex, in S. cerevisiae.

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene.

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

A novel role for thioredoxin reductase in the iron metabolism of S. cerevisiae.

Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.

Efficacy and tissue distribution of DHP107, an oral paclitaxel formulation.

Paclitaxel is indispensable in treating human cancers. Due to poor drug solubility and efflux systems in the gastrointestinal tract, peroral delivery of paclitaxel has been a significant challenge. We developed a mucoadhesive oral formulation (DHP107) that can directly and effectively deliver paclitaxel to intestinal endothelial cells without concomitant use of P-glycoprotein inhibitors. Here, we evaluated the tissue distribution of paclitaxel, the antitumor efficacy and the absorption mechanism of DHP107. DHP107, which contains 10 mg/mL of paclitaxel in a mixture of monoolein, tricarprylin, and Tween 80 was administered p.o. to female BALB/c mice at a 50 mg/kg dose. Diluted Taxol was administered via bolus tail-vein injection at 10 mg/kg as a control. Blood and tissue samples were harvested at various time points and analyzed by high-performance liquid chromatography. Tissue sections were observed using light microscopy after immunohistochemical and Oil Red O staining. By day 27, tumor volume after DHP107 and Taxol treatments was one-third of that in the untreated group. After p.o. administration, paclitaxel was widely distributed in various organs (T(max) = 2 h), especially liver, spleen, and lung. DHP107 was effectively absorbed through the intestinal lipid transport system. DHP107 changed spontaneously into <100-mum droplets and micelles in the intestine, which in turn adhered to mucoepithelial cells, were absorbed via lipid uptake mechanism, and formed lipid bodies in the epithelium. Paclitaxel in DHP107 was effectively absorbed through the gastrointestinal tract via lipid uptake mechanism and was distributed in various tissues. The detailed uptake mechanism is currently under investigation.

New and efficient method using Saccharomyces cerevisiae mutants for identification of siderophores produced by microorganisms.

The separation and identification of siderophores produced by microorganisms is a time-consuming and an expensive procedure. We have developed a new and efficient method to identify siderophores using well-established Saccharomyces cerevisiae deletion mutants. The Deltafet3,arn strains fail to sustain growth, even when specific siderophores are supplied, and mutants are siderophore-specific: Deltafet3,arn2 for triacetylfusarinine C (TAFC), Deltafet3,arn1,sit1 for ferrichrome (FC), and Deltafet3,sit1 for ferrioxamine B (FOB). The culture broth of Fusarium graminearum was separated by HPLC, and each peak was subjected to a plate assay using S. cerevisiae mutants. We have found that each peak contained specific siderophores produced by F. graminearum, and these coincided with reference siderophores. This method is quite novel because nobody tried this method to identify the siderophores. Furthermore, this method will save time and cost in the identification of siderophores produced by microorganisms.

Physical and functional interaction of FgFtr1-FgFet1 and FgFtr2-FgFet2 is required for iron uptake in Fusarium graminearum.

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.

Functional characterization of Hsp33 protein from Bacillus psychrosaccharolyticus; additional function of HSP33 on resistance to solvent stress.

Psychrophiles have been known as efficient organism to degrade organic solvent. To investigate the mechanism of solvent stress and identify the factors that affect the solvent stress in psychrophiles, we selected Bacillus psychrosaccharolyticus one of the psychrophiles and two-dimensional gel electrophoresis was performed. Among the protein spots analyzed by 2-DE, five spots induced in 3% IPA stress conditions were identified by MS/MS, and one of these spots was identified as a Hsp33 family. The Hsp33 protein sequence of B. psychrosaccharolyticus exhibited a high similarity with the corresponding proteins of other bacteria. The Hsp33 protein of B. psychrosaccharolyticus has a highly conserved zinc-binding domain (CXCX, CXXC) that includes four cysteine residues in the C-terminus. In addition, the transcriptional induction of the HSP33 of B. psychrosaccharolyticus was confirmed by Northern blot analysis, and formation of free thiol linkage was induced under stress conditions such as exposure to solvents, heat-shock, and oxidative stress. Furthermore, over-expressed strains of HSP33 of B. psychrosaccharolyticus in Escherichia coli improved stress tolerance to the organic solvent when compared with the wild-type. These data suggest that the solvent stress condition was similar to heat-shock or oxidative stress, especially through the triggering of induction and activation of a redox-regulatory chaperone, Hsp33, and Hsp33 plays a critical role in the tolerance to stress.

Induction of immunity against hepatitis B virus surface antigen by intranasal DNA vaccination using a cationic emulsion as a mucosal gene carrier.

Delivery of DNA vaccines to airway mucosa would be an ideal method for mucosal immunization. However, there have been few reports of a suitable gene delivery system. In this study we used a cationic emulsion to immunize mice via the intranasal route with pCMV-S coding for Hepatitis B virus surface antigen (HBsAg). Complexing pCMV-S with a cationic emulsion dramatically enhanced HBsAg expression in both nasal tissue and lung, and was associated with increases in the levels of HBs-specific Abs in serum and mucosal fluids, of cytotoxic T lymphocytes (CTL) in the spleen and cervical and iliac lymph nodes, and of delayed-type hypersensitivity (DTH) against HBsAg. In contrast, very weak humoral and cellular immunities were observed following immunization with naked DNA. In support of these observations, a higher proliferative response of spleenocytes was detected in the group immunized with the emulsion/pCMV-S complex than in the group immunized with naked pCMV-S. These findings may facilitate development of an emulsion-mediated gene vaccination technique for use against intracellular pathogens that invade mucosal surfaces.

Cellular iron utilization is regulated by putative siderophore transporter FgSit1 not by free iron transporter in Fusarium graminearum.

This report investigated FgSit1, which encodes a putative ferrichrome transporter of Fusarium graminearum. The identity of the deduced amino acid sequence of FgSit1 with the amino acid sequence of ScArn1p, an FC-Fe(3+) transporter of Saccharomyces cerevisiae, was 51%; both the growth defect related to the Deltafet3Deltaarn1-4 strain of S. cerevisiae in an iron-depleted condition and the FC-Fe(3+) uptake activity were recovered upon the introduction of FgSit1 into the Deltafet3Deltaarn1-4 strain. Although ScArn1p was found in the late endosomal compartment in S. cerevisiae, FgSit1 was found on the plasma membrane in S. cerevisiae; when FgSit1 was expressed exogenously in S. cerevisiae, it showed greater FC-Fe(3+) uptake activity than did ScArn1p. Additionally, in F. graminearum FC-Fe(3+) uptake activity in the Deltafgsit1 strain was found to be one-fourth that of the wild-type. However, Fe(3+) uptake activity in the Deltafgsit1 strain was 5-fold higher than that of wild-type; the gene expression of FgFtr1, a putative iron transporter, was induced by the deletion of FgSit1, but was not induced by the deletion of FgSit2. Taken together, these results strongly suggest that FgSit1 encodes a putative FC-Fe(3+) transporter that mediates FC-Fe(3+) uptake using a different mechanism than ScArn1p and plays an important role in the regulation of cellular iron availability in F. graminearum.

The novel antioxidant 3-O-caffeoyl-1-methylquinic acid induces Nrf2-dependent phase II detoxifying genes and alters intracellular glutathione redox.

Induction of detoxifying phase II genes by chemopreventive agents represents a coordinated protective response against oxidative stress and neoplastic effects of carcinogens. We have earlier shown that a novel antioxidant from the bamboo leaves constituent 3-O-caffeoyl-1-methylquinic acid (MCGA3) induces heme oxygenase-1 (HO-1) and protects endothelial cells from ROS-induced endothelial injury. The purpose of this study was to elucidate the induction mechanism of HO-1 and other phase II genes by MCGA3 in human umbilical vascular endothelial cells (HUVECs). Using Northern blotting and RT-PCR, we found that treatment of HUVECs with MCGA3 increased, in a dose and time-dependent manner, steady-state mRNA levels of the selected phase II genes including HO-1, ferritin, gamma-glutamylcysteine lygase, glutathione reductase, and glutathione transferase, which were dependent on Nrf2 nuclear translocation. The observed phase II gene induction by MCGA3 was found to be associated with MCGA3-mediated cytoprotective activity, ROS-scavenging potency, and the increase in the cellular levels of both reduced (GSH) and oxidized glutathione (GSSG). Interestingly, exposure to MCGA3 resulted in a decreased ratio of GSH/GSSG, which was negatively related with mRNA level of phase II genes. By employing N-acetylcysteine and GSH biosynthetic enzyme inhibitors as well as prooxidants, hemin and H(2)O(2), we show that a decreased intracellular GSH/GSSG homeostasis, at least in part, may be involved in the MCGA3-mediated phase II gene induction and Nrf2 translocation, although the attenuation of HO-1 expression with SP 600125 supports a partial involvement of JNK signaling.

Functional identification of high-affinity iron permeases from Fusarium graminearum.

The ScFTR1 gene encodes an iron permease in Saccharomyces cerevisiae. Its homologues, FgFtr1 and FgFtr2, were identified from filamentous pathogenic plant fungus, Fusarium graminearum. Homologies between the deduced amino acid sequences of ScFtr1p and FgFtr1 and FgFtr2 were 56 and 54%, respectively, and both had REXXE sequences, which form the conserved amino acid sequence of ScFtr1p. FgFtr1 expression increased under iron depletion, and although FgFtr2 mRNA was not detected in the wild-type strain, it was detected in the deltafgftr1 strain in the iron-depleted condition. When the FgFtr1 and FgFtr2 were deleted, the amount of growth was found not to be different from the wild-type in iron-depleted media. However, the mRNA of FgSid, a homologue of the SIDA of Aspergillus fumigatus, was dramatically increased in the deltafgftr1/deltafgftr2 strain and in an iron-depleted condition. FgFtr1 and FgFtr2 genes act as functional complements when they are introduced into the S. cerevisiae deltaScftr1 strain. The deltaScftr1 strain, which contains either the FgFtr1 or FgFtr2, grew well in iron-depleted media. Moreover, specific alteration of the REXXE consensus sequence of FgFtr1 and FgFtr2 did not allow for sustained growth of the deltaScftr1 strain on iron-depleted medium. The iron uptake activity was recovered when FgFtr1 and FgFtr2 genes were introduced into the deltaScftr1 strain. Though the Fet3p in S. cerevisiae was found on the intracellular vesicle in the deltaScftr1 strain, Fet3p was found on the plasma membrane when FgFtr1 or FgFtr2 was introduced into the deltaftr1 strain. An infection test was carried out with deletion strains; however, no change in the ability of these strains to cause disease was observed. These results suggest that FgFtr1 and FgFtr2 may function as iron permeases in the reductive iron uptake pathway and that they do not play major roles in the pathogenicity of F. graminearum.

Airway gene transfer using cationic emulsion as a mucosal gene carrier.

BACKGROUND: Delivery of genes to airway mucosa would be a very valuable method for gene therapy and vaccination. However, there have been few reports on suitable gene delivery systems for administration. In this study, we use a cationic emulsion system, which is physically stable and facilitates the transfer of genes in the presence of up to 90% serum, as a mucosal gene carrier. METHODS AND RESULTS: Cationic lipid emulsion was formulated with squalene and 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP) as major components. Emulsions formed stable complexes with DNA and protected and transferred DNA to target cells against DNase I digestion in the presence of mucosal destabilizers such as heparin sulfate (a polysaccharide of the glycosaminoglycan family in mucosa) and Newfectan (a natural lung extract of bovine) in an in vitro system. In contrast, commercial liposomes and counter liposomes, made with an identical lipid composition of emulsions, failed. After in vivo intranasal instillation, the cationic emulsion showed at least 200 times better transfection activity than the liposomal carriers in both nasal tissue and lung. CONCLUSIONS: These findings show that cationic emulsions can mediate gene transfection into airway epithelium, making it a good choice for transferring therapeutic genes and for genetic vaccination against an pathogenic infection via an airway route.

Polycations enhance emulsion-mediated in vitro and in vivo transfection.

To enhance the in vitro and in vivo transfection activity of the cationic lipid emulsion (LE), three natural polycations, protamine sulfate (PS), poly-L-lysine and spermine, were selected as DNA condensing active agents. Formation of the LE/polycation/DNA ternary complexes was identified by using agarose gel retardation study. The structure of these complexes was characterized by measuring the complex size and the decrease of the DNA fluorescence in the presence of ethidium bromide (EtBr). By adding a polycation, the particle size of the complex decreased, and DNA in the complex became highly condensed and resistant to intercalation of EtBr. Among the polycations, PS yielded the most highly compacted ternary complex. In vitro and in vivo transfection activities of the complexes were determined using various cell lines and Balb/c mouse intravenously and intranasally, respectively. The transfection activity of the ternary complex increases by at least 2.5-5-fold in vitro cell culture system in the presence of 80% serum as well as in vivo mouse system, as compared with LE/DNA binary complexes. More importantly, after intravenous and intranasal administrations, the in vivo transfection efficiency of the LE/PS/DNA complex was ca. 30 and 50 times higher than that of the liposome (LP)/DNA complex in spleen and lung, respectively. On the other hand, cell toxicity of the ternary complex is lower than that of binary complex. Thus, we conclude that the pre-condensation of DNA with polycations can be a promising approach to further increase in vitro and in vivo transfection efficiency of cationic lipid emulsion.

Sed1p interacts with Arn3p physically and mediates ferrioxamine B uptake in Saccharomyces cerevisiae.

Two-hybrid analysis can be used to study protein function and metabolic pathways. Using yeast two-hybrid analysis to identify a siderophore uptake pathway in the yeast Saccharomyces cerevisiae, we found that the C-terminal part of the cell-wall protein Sed1p interacts with the N-terminal region of Arn3p. To confirm the physical interaction between the Sed1p C-terminal fragment and the hydrophilic N-terminal fragment of Arn3p, we used an in vitro co-immunoprecipitation assay and a growth test of the strain with bait and SED1 plasmids in quadruple amino acid-depleted medium. The expression of SED1 was upregulated by overexpression of AFT1-1(up) under the control of the GAL promoter. This occurred despite the lack of an Aft1p-binding consensus region on the upstream region of SED1 or a high concentration of free iron. Although free-iron uptake activity in the Deltased1 strain did not differ from that in the parental strain, ferrioxamine bound-iron uptake activity was reduced in the Deltased1 strain. Moreover, the Deltased1 strain showed low viability at high iron concentrations. Taken together, these results suggest that Sed1p mediates siderophore transport and confers iron resistance in S. cerevisiae.

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.

Cytoprotective effects of heme oxygenase-1 induction by 3-O-caffeoyl-1-methylquinic acid.

The novel antioxidant 3-O-caffeoyl-one-methylquinic acid (MCGA3) is a methyl chlorogenic acid derivative isolated from bamboo leaves. MCGA3 scavenges reactive oxygen species (ROS) and inhibits lipid peroxidation and xanthine oxidase in vitro. In this study, we evaluated the cytoprotective effect of MCGA3, which occurs via heme oxygenase-1 (HO-1) induction in bovine vascular endothelial cells exposed to tert-butylhydroperoxide (tBHP). Cells treated with 1 mM tBHP (6-18 h) generated substantial ROS and concomitantly lost most intracellular lactate dehydrogenase (LDH), which then caused necrotic cell death. Of the several MCGA antioxidants and structurally related phenolic acids examined in this study, MCGA3 (0.01-0.15 mM) was found to completely block this necrosis and generation of ROS by tBHP. Surprisingly, MCGA3 by itself was found to be a potent inducer of HO-1. We observed the time- and dose-dependent induction of HO-1 mRNA and protein, which was closely associated with decreased intracellular ROS and necrosis against tBHP. Deesterified or Al-chelated MCGA3 or co-treatment with MCGA3 and actinomycin D abolished HO-1 induction and the antinecrotic effect of MCGA3. Zinc protoporphyrin IX and cycloheximide attenuated the cytoprotection afforded by MCGA3, but did not reduce HO-1 mRNA. Interestingly, N-acetylcysteine (1 mM) enhanced the HO-1 induction of MCGA3, but N-acetylcysteine itself did not induce HO-1. These results suggested that not only ortho-dihydroxyl groups but also aromatic ester and methoxyl ester moieties are necessary for full HO-1 induction and cytoprotection against toxic tBHP-derived ROS. Ferritin mRNA was also upregulated during all HO-1 induction by MCGA3, which might decrease iron and lower ROS levels. Consequently, the combined action of HO-1 and ferritin may protect cells from toxic tBHP-mediated necrosis.

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Use of acetate for enrichment of electrochemically active microorganisms and their 16S rDNA analyses.

A fuel cell-type electrochemical device has been used to enrich microbes oxidizing acetate with concomitant electricity generation without using an electron mediator from activated sludge. The device generated a stable current of around 5 mA with complete oxidation of 5 mM acetate at the hydraulic retention time of 2.5 h after 4 weeks of enrichment. Over 70% of electrons available from acetate oxidation was recovered as current. Carbon monoxide or hydrogen did not influence acetate oxidation or current generation from the microbial fuel cell (MFC). Denaturing gradient gel electrophoresis showed that DNA extracted from the acetate-enriched MFC had different 16S rDNA patterns from those of sludge or glucose+glutamate-enriched MFCs. Nearly complete 16S rDNA sequence analyses showed that diverse bacteria were enriched in the MFC fed with acetate. Electron microscopic observations showed biofilm developed on the electrode, but not microbial clumps observed in MFCs fed with complex fuel such as glucose and wastewater from a corn-processing factory.

Expression and purification of the anticomplementary peptide Sh-CRIT-ed1 (formerly Sh-TOR-ed1) as a tetramultimer in Escherichia coli.

Many complement inhibitors found in plants and other organisms have been recognized as an antiinflammatory drug. Sh-CRIT-ed1 is a complement inhibitory peptide, present on the Schistosoma parasite surface. In the present study, we expressed chemically synthesized oligonucleotides encoding Sh-CRIT-ed1 with an additional hexahistidine codon at the C-terminus and purified in Escherichia coli BL21. The cloned gene, which was multimerized four times in pBlue-script II KS (+) at the isoschizomer sites (BamHI, BglII), was named Sh4, and expressed in E. coli BL21 harboring pGEX-KG. The fusion protein (GST-Sh4) was purified with high yield successively by affinity chromatographies of glutathione-Sepharose 4B and Ni-NTA-agarose. Recombinant Sh-CRIT-ed1 was obtained readily by thrombin digestion and CNBr cleavage of GST-Sh4, and the yield was 9.03 mg from 1-liter culture of E. coli BL21 harboring pGEX-Sh4. The recombinant Sh-CRIT-ed1 showed strong anticomplementary activity (IC(50) = 6.02 microM) by complement haemolysis assay.

Isolation and characterization of anticomplementary beta-glucans from the shoots of bamboo Phyllostachys edulis.

Bamboo, Phyllostachys edulis produces well known edible shoots in Asia. Immunostimulating anticomplementary (complement activating) substances have been recognized as a characteristic biological response modifier (BRM). In the present study, we isolated and characterized three anticomplementary beta-glucans (BS-BGA, BS-BGB, and BS-BGC) from bamboo shoots. Hot-water extraction, DEAE-Toyopearl 650M-column chromatography, amylase digestion and concanavalin A adsorption, and Sephacryl S-100 HR column chromatography were applied to isolate the beta-glucans. The average molecular masses of the beta-glucans were estimated to be from 14,500 to 85,300 Da by HPSEC-MALLS-RI. All three beta-glucans (0.1-1.0 mg/mL) activated the complement system via the alternative pathway, and could cleave human complement C3 under Ca 2+-free gelatin veronal buffered saline. Among them, the largest molecule, BS-BGA was the most potent complement activator. Methylation analysis and NMR spectroscopy were used to achieve their structural characterization. They are all water-soluble and composed mainly of backbone structures of beta-(1-->3)-glucan with beta-(1-->4)-linked side chains varying in degree of branching. BS-BGA consisted of a higher proportion of 3-linked glucopyranosyl residues and a lower degree of branching than BS-BGB and BS-BGC. In particular, BS-BGA contained a small amount of O-acetyl groups at C-6 of the 3-linked glucopyranosyl residues. These data demonstrate that the structural characteristics including molecular size, degree of branching, and O-acetyl substitution are involved, at least in part, in their different anticomplementary activities.