Intergeneric conjugal transfer of plasmid DNA from<Emphasis Type="Italic"> Escherichia coli</Emphasis> to<Emphasis Type="Italic"> Kitasatospora setae</Emphasis>, a bafilomycin B<Subscript>1</Subscript> producer

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1×10^-6) was achieved on ISP4 medium containing 30 mM MgCl₂ using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non-Streptomyces actinomycetes. K. setae contains a single C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.     

Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.     

Comparative effect of repeated ingestion of difructose anhydride III and palatinose on the induction of gastrointestinal symptoms in humans

We evaluated the safety and change in fermentability from repeated ingestion of difructose anhydride III (DFAIII) in humans. A randomized controlled single-blind crossover study with thirteen subjects was conducted. Each subject ingested 5 g of DFAIII or palatinose daily for 12 days, before and after which the subject was loaded with 10 g of DFAIII and had breath hydrogen measured from 0 to 9 h (DL test) to evaluate the fermentability of DFAIII. The defecation frequency and abdominal symptom score were the same between each ingestion period. Moreover, DFAIII ingestion had no influence on blood test results. Only the breath hydrogen excretion in post-DFAIII ingestion was slightly higher at h 8 than the pre-ingestion. Consequently, repeated ingestion of DFAIII for 12 days was as safe as palatinose ingestion, especially with respect to abdominal symptoms and blood test results, and its high resistance to enterobacterial fermentation in humans was not impaired.     

Effects of a novel gaseous antioxidative system containing a rosemary extract on the oxidation induced by nitrogen dioxide and ultraviolet radiation

Rosemary is commonly used as a spice and a flavoring agent in food processing. Although the antioxidative properties of its extracts have been investigated, there have been few reports on the volatile components of rosemary. We designed a novel antioxidative system which can generate the volatile constituents in the gaseous phase from a rosemary extract and evaluated the gaseous antioxidative activities against both lipid peroxidation and cell death induced by nitrogen dioxide and ultraviolet radiation. The antioxidative effects of the major volatile components on the oxidation of linoleic acid induced by azo compounds were also investigated in a solution. The volatile components in the novel antioxidative system suppressed the Jurkat cell death induced by nitrogen dioxide and the intracellular formation of reactive oxygen species in fibroblast cells induced by ultraviolet radiation. 1,8-Cineole among the volatile components exerted an antioxidative effect against the oxidation of linoleic acid in a solution induced by azo compounds and ultraviolet radiation. These data suggest that the volatile constituents of a rosemary extract had antioxidative properties and that gaseous exposure antioxidant is a promising method for promoting health.     

3alpha/beta,20beta-hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH

Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.     

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa ). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH(4)(+) as the amino donor. The V (max) and K (i) for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

Detection of Pairwise Residue Proximity by Covariation Analysis for 3D-Structure Prediction of G-Protein-Coupled Receptors

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.