Distribution of microbes producing antimicrobial epsilon-poly-L-lysine polymers in soil microflora determined by a novel method

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), epsilon-poly-L-lysine (epsilon-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected epsilon-PL producers by electrostatic interaction with the secreted basic polymers. All epsilon-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family Streptomycetaceae and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of epsilon-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an epsilon-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.     

Isolation of a new Pseudomonas halophila strain possess bacteriorhodopsin-like protein by a novel method for screening of photoactive protein producing bacteria

Bacteriorhodopsin (bR) is a transmembrane protein deposited in the purple membrane of Halobacterium salinarum which absorbs energy from photons to create a photo-induced proton gradient across the membrane. A bR molecule can be considered as a natural solar device transforming light into other types of energy and therefore is of interest for a wide range of applications including two and three-dimensional memory storage, optical data processing, artificial cells, holographic media, the artificial retina and photo sensor devices. H. salinarum is a slow-growing, halophilic Archaea present in red salt waters. The present study introduces a novel bR-like pigment from a new strain of Pseudomonas halophila (with registered accession number KC959570 in the NCBI databank) which has a very significant degree of light-dependent activity. This is the first report on the presence of functional bR-like protein in the Pseudomonas family. The isolate is a fast-growing, halophilic bacterium and is comparable with other photoactive protein producer microorganisms. Also, in the present study a novel isolation method for screen light-stimulating protein producing microorganisms is introduced. For this purpose 2,3,5-triphenyltetrazolium chloride (TTC) was employed for the first time as an artificial hydrogen acceptor in the proton-transfer processes. The TTC test is an easy and susceptible method for estimating hydrogen production during the proton transport process. This is the first report of the use of TTC for photo activity measurement and selection of bacteria containing light dependent proteins.     

化学-酶法制备L-高苯丙氨酸

以苯丙酸乙酯为原料,通过正交设计优化2-氧4-苯基丁酸盐的制备条件：苯丙酸乙酯与草酸二乙酯摩尔比为1：3,缩合反应时间为2．5h,H2SO4质量分数为20％,水解反应时间为15h,优化条件下2-氧-4-苯基丁酸盐的产率为68．24％。随后,利用E．coli A5所产的天冬氨酸转氨酶为生物催化剂制备L-高笨丙氨酸。酶转化反应的最适条件为：游离细胞体系pH、温度、底物质量浓度和细胞质量浓度分别为8．5、37℃、20g/L和30g／L;而固定化细胞体系则分别为7．0—9．0、40℃、10g／L和30g/L。采用廉价的L-谷氨酸（L—Glu）作为氨基供体,添加表面活性剂有利于提高L-HPA产率。通过研究固定化细胞转化反应进程,结果发现8h内90％的底物可转化为L—HPA。     

A novel method for producing a foodstuff from defatted black sesame seed that inhibits allergen absorption

A method is proposed to produce a foodstuff that inhibits allergen absorption through the intestinal tract. Defatted black sesame (Sesamum indicum) seeds as a starting material were hydrolyzed with a crude preparation of trypsin at 40 degrees C and pH 8 for 3 hrs while gently stirring to generate an active peptide. The resulting hydrolysate was heated to inactivate the trypsin and make the active components soluble. An extract was obtained by centrifugation and then freeze-dried. Ser-Asn-Ala-Leu-Val-Ser-Pro-Asp-Trp-Ser-Met-Thr-Gly-His (compound 1) as an active peptide, and sesamino1 2'-O-beta-glucopyranosyl-(1-->2)-O-[beta-glucopyranosyl(1-->6)]-O-beta-glucopyranoside (compound 2) and sesamino1 2'-O-beta-glucopyranosyl (1-->2)-O-beta-glucopyranoside (compound 3) were identified as active lignan glycosides in an in vitro model by using Caco-2 cells. Compound 1 was active at 10(-7) M and compounds 2 and 3 at 10(-5) M.     

A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

A novel protein refolding method using a zeolite

We have succeeded in developing a simple and effective protein refolding method using the inorganic catalyst, beta-zeolite. The method involves the adsorption of proteins solubilized with 6M guanidine hydrochloride from inclusion body (IB) preparations onto the zeolite. The denaturant is then removed, and the proteins in the IBs are released from the zeolite with polyoxyethylene detergent and salt. All of the IBs tested (11 different species) were successfully refolded under these conditions. The refolded proteins are biochemically active, and NMR analysis of one of the proteins (replication protein A 8) supports the conclusion that correct refolding does occur. Based on these results, we discuss the refolding mechanism.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

A novel electroporation method using a capillary and wire-type electrode

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.     

Application of a novel protein biochip technology for detection and identification of rheumatoid arthritis biomarkers in synovial fluid

We compared protein profiles of the synovial fluid of patients with rheumatoid arthritis and osteoarthritis by using surface-enhanced laser desorption/ionization mass spectrometry technology. With this approach, we identified a protein expressed specifically in the synovial fluid of the patients with rheumatoid arthritis. During the investigation, we found several reproducible and discriminatory biomarker candidates for distinction between rheumatoid arthritis and osteoarthritis. Among these candidates, a 10 850 Da protein peak was the clearest example of a single signal found specifically in the rheumatoid arthritis samples. This candidate was purified using a size-exclusion spin column followed by gel electrophoresis and subsequently identified by peptide mapping and post-source decay (PSD) analysis. The results clearly indicate that the protein is myeloid-related protein 8, which was verified by the enzyme immunoassay. It is known that the myeloid-related protein 8 level in serum and synovial fluid is related to disease activity in juvenile rheumatoid arthritis. The results suggest that the ProteinChip platform is useful to detect and identify protein biomarkers expressed specifically in diseases or in some stage of diseases.     

A novel strategy for producing chimeric bispecific antibodies by gene transfection

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation. Transfectomas secreting bispecific antibodies were identified. This approach has advantages over the fusion of hybridomas or chemical linking of two antibody molecules and will enable the use of bispecific antibodies in vivo.     

PathAct: a novel method for pathway analysis using gene expression profiles

We developed PathAct, a novel method for pathway analysis to investigate the biological and clinical implications of the gene expression profiles. The advantage of PathAct in comparison with the conventional pathway analysis methods is that it can estimate pathway activity levels for individual patient quantitatively in the form of a pathway-by-sample matrix. This matrix can be used for further analysis such as hierarchical clustering and other analysis methods. To evaluate the feasibility of PathAct, comparison with frequently used gene-enrichment analysis methods was conducted using two public microarray datasets. The dataset #1 was that of breast cancer patients, and we investigated pathways associated with triple-negative breast cancer by PathAct, compared with those obtained by gene set enrichment analysis (GSEA). The dataset #2 was another breast cancer dataset with disease-free survival (DFS) of each patient. Contribution by each pathway to prognosis was investigated by our method as well as the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis. In the dataset #1, four out of the six pathways that satisfied p < 0.05 and FDR < 0.30 by GSEA were also included in those obtained by the PathAct method. For the dataset #2, two pathways (“Cell Cycle” and “DNA replication”) out of four pathways by PathAct were commonly identified by DAVID analysis. Thus, we confirmed a good degree of agreement among PathAct and conventional methods. Moreover, several applications of further statistical analyses such as hierarchical cluster analysis by pathway activity, correlation analysis and survival analysis between pathways were conducted.     

Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye

The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV.     

A novel method of producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute enzyme

(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharmaceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis and bioconversion. Bioconversion in this study is a key reaction, since optically active carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonylsuccinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonylamino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells. Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme and therefore, may be of interest for future industrial applications.     

A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

Background  

Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with ⁵¹chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry.     

Analysis of characters coded for by T-DNA using hybridization between tumorous and normal plant cells

In somatic hybrids between tumourous Nicotiana tabacum (B6S3) and normal mesophyll Atropa belladonna cells, the following traits, directly or indirectly connected with T-DNA gene expression and tumourous growth, were analysed: lysopine dehydrogenase activity (LpDH), shoot suppression, root suppression, ability to grow on media with D-lactose as a sole carbon source and resistance to 2-aminoethylcysteine, 5-bromodeoxyuridine and 5-methyltryptophan. Dominant (semidominant) expression was observed for all but one trait studied, e.g. shoot suppression which behaved as a recessive character.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - NAA Naphtaleneacetic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - 5-BUdR 5-Bromodeoxyuridine - 2-AEC 2-Aminoethylcysteine - 5-MT 5-Methyltryptophan     

Validity and reliability of a visual scoring method for masticatory ability using test gummy jelly

Background: For quantitative evaluation of masticatory ability of the elderly patients, there should be a simple and reliable method without special techniques and instruments. Objective: The purpose of this study was to examine the validity and reliability of a visual scoring method for assessing masticatory performance. Materials and Methods: A 10‐stage scale for visually scoring was rated based on the range of the glucose concentration dissolved from comminuted jelly. Photographic images of comminuted jellies were produced as a standard material for each score. Fifty subjects were recruited as raters who graded the visual score for 50 photographic images of comminuted jellies on the screen of a lap‐top three times in random order. Results: There were strong correlations (rs = 0.911– 0.981, Spearman’s rank coefficient) between the actual scores determined from the glucose concentration and the visual scores graded by subjects in all three measurements. The intraclass correlation coefficients (ICCs) of the inter‐rater reliability and the ICCs of the intra‐rater reliability of the visual scoring ranged from 0.946 to 0.947 and from 0.860 to 0.987 in three measurements, respectively. Conclusions: These results indicated that the visual scoring method was valid and reliable for evaluation of masticatory performance.     

Isolation and cultivation of a novel microorganism producing a maltopentaose-forming enzyme

Summary A novel microorganism producing a maltopentaose-forming enzyme was screened from a soil sample. The enzyme produced by the bacteria formed maltopentaose from starch during the first stage of the reaction. The bacterium was characterized asPseudomonas sp. (KO-8940) on the basis of its morphological, physiological and biological properties. Culture conditions for enzyme production were investigated and established. The medium was composed of beef extract 0.8%, ammonium sulphate 1.0% and maltose 0.8% in tap water. Optimum conditions for bacterial growth were: initial pH 8.0, temperature 45°C, and 3 days cultivation with a rotary shaker (200 rpm). The broth supernatant obtained contained the maltopentaose-forming crude enzyme, with which 40% of starch was converted to maltopentaose.