PubMed Health⌕ Search

Biomedical subjects

Hiroshi Saiki

Publications and source records attributed to Hiroshi Saiki.

18 recordsLinked to original sources
PubMed2006

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay.

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.

Biosensing Techniques↗
PubMed2006

Acidianus manzaensis sp. nov., a novel thermoacidophilic archaeon growing autotrophically by the oxidation of H2 with the reduction of Fe3+.

A novel thermoacidophilic iron-reducing Archaeon, strain NA-1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H2 or S0 as an electron donor and Fe3+ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe3+ and O2 served as electron acceptors for growth. However, S0, NO3-, NO2-, SO4(2-), Mn4+, fumarate, and Fe2O3 did not serve as electron acceptors. The ranges of growth temperature and pH were 60-90 degrees C (optimum: 80 degrees C) and pH 1.0-5.0 (optimum: pH 1.2-1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1T = NBRC 100595 = ATCC BAA 1057).

Acidianus↗
PubMed2006

Improving an immunoassay response to related polychlorinated biphenyl analytes by mixing antibodies.

Immunoassays for detection of a class of closely related antigens, e.g., PCBs, have often been too specific (responding strongly to some members of the class and missing others) and no general method for adjusting the response has been described. In this paper, the difference in the response of a model immunoassay to different Kanechlors (Japanese commercial mixtures of PCBs, analogous to Aroclors in the United States) is reduced from 20- or 50-fold (depending on which antibody is used) to 3-fold when the antibodies are mixed at the proper ratio. A mathematical model based on competitive binding of two antibodies for up to four antigens has been developed and used to describe the assay performance and to predict optimum mix ratios for the antibodies used. The model (based on separate measurement of each antibody's effective Kd for each Kanechlor) provides an excellent fit to the measured mixed antibody assay response. The model is also successful in identifying cases where mixing monoclonal antibodies will not improve the response. It is thought the method described will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

Antibodies↗
PubMed2005

Symbiotic association in Chlorella culture.

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.

Bacteria↗
PubMed2004

Evaluation of a compact bench top immunoassay analyzer for automatic and near continuous monitoring of a sample for environmental contaminants.

A compact bench top immunoassay analyzer is evaluated and shown to possess sufficient automation to allow continuous unattended sampling and measuring while still achieving the theoretical (antibody affinity based) detection limit for analyte. The system is comprised of antigen coated particles in a disposable flow cell held at the focus of a filter fluorometer. Capture of fluorescently labeled antibody from the flow stream is inhibited by analyte in the sample, allowing analyte concentrations to be determined from the fluorescent intensity. The disposable cell was designed to allow easy end user changing of test specificity, e.g. for selection of any member of a panel of environmental contaminants. Standard curves are shown for six analytes of environmental interest, dioxin F114 (2,3,4,7,8-PeCDF), the pesticide Fenitrothion, three coplanar PCBs, including the most toxic, PCB 126, and estradiol. In each case the curves are constructed using antibody concentrations at or below the Kd of the antibody, assuring that the sensitivity shown is limited by the antibody itself rather than the analyzer. The dynamic range for the six analytes investigated ranged from a low of 5 to 340 pM for fenitrothion to a high of 0.8 to 59 nM for dioxin F114, and is correlated to the antibody Kd in every case. Data is also shown for 17 consecutive samples, including both high and low values, measured completely automatically over a period of hours. With further development and characterization, the bench top analyzer is expected to fill an important niche in environmental testing.

Biosensing Techniques↗
PubMed2004

A combination of labeled and unlabeled antibody enables self-calibration and reduction of sample matrix effects in immunoassay.

Sample matrices interfering with analyte determinations, termed matrix effects, are one of the factors limiting the more widespread use of environmental immunoassays. Previous attempts to reduce matrix effects have focused on particular assays in specific matrices rather than on general methods. Here we describe a novel method to eliminate one class of matrix effects in immunoassay, independent of the particular matrix or analyte. The method is demonstrated with a model system detecting estradiol in either a 10% methanol or a 5% dimethyl sulfoxide matrix. Fluorescently labeled antiestradiol antibody is introduced as the detecting antibody and excess unlabeled antiestradiol antibody is included as a reference antibody. The binding of the excess reference antibody to the sample analyte artificially creates a sample containing no free analyte to bind to the detecting antibody. This allows estimation of the fluorescent signal for "zero" analyte in the actual sample matrix. The solvents employed as model systems reduce the affinity of the detecting antibody and cause false positive results at low estradiol concentrations and false negative results at high concentrations. The proposed reference method, including addition of the reference antibody, resulted in a self-calibrating assay in which the matrix effects, both positive and negative, were completely eliminated.

Antibodies, Monoclonal↗
PubMed2004

Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects.

A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.

Antibodies, Monoclonal↗
PubMed2004

Analysis of gene expression in yeast protoplasts using DNA microarrays and their application for efficient production of invertase and alpha-glucosidase.

The global gene expression of cultured Saccharomyces cerevisiae protoplasts was compared with that of cells using DNA microarray. Quantitative and qualitative analyses revealed that after 6 h of cultivation, 416 gene transcript levels (about 7.1% in all) in the cultured protoplasts were different from those in the cells. Various characteristics and functions of the protoplasts were predicted from the analysis of the gene functions. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed two novel methods for the production of useful enzymes based on the characteristics of protoplasts. One was the production of invertase based on the activated secretory pathway, while the other was the production of alpha-glucosidase based on the activated carbohydrate metabolism. The secretion of invertase and alpha-glucosidase was promoted in cultured protoplasts. The invertase and alpha-glucosidase productivities in the cultured protoplasts were 657 U and 218 U, respectively. On the other hand, only 227 U of invertase was produced, while alpha-glucosidase was not detected, in the cultured cells. The fragile protoplasts were immobilized in agarose gel to protect them from hydrodynamic stress. Four repeated-batch cultures with the immobilized protoplasts were performed, leading to the production of 1574 U of invertase and 739 U of alpha-glucosidase. The same productivities were obtained when this system was scaled up by 10-fold (invertase: 13304 U; alpha-glucosidase: 7688 U).

Journal Article↗
PubMed2003

Combinational use of antibody affinities in an immunoassay for extension of dynamic range and detection of multiple analytes.

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.

Antibodies↗
PubMed2003

Respiratory isozyme, two types of rusticyanin of Acidithiobacillus ferrooxidans.

Among the members of the copper protein superfamily, the type I enzyme rusticyanin, which is found as an electron carrier in the oxidative respiratory chain of Acidithiobacillus ferrooxidans, is the only one to have both a high redox potential and acid stability. Here we report that two forms of the rusticyanin gene (rus) are present in the genomes of some strains of A. ferrooxidans. The more common form of rus (type-A) was found to be present in all six strains studied, including those harboring only a single copy of the gene. In addition a less common form (type-B) occurred in strains harboring multiple copies of the gene. The two genes were expressed as rusticyanin isozymes with differing surface charges due to differences in their amino acid composition. Still, the copper coordination sites were completely conserved, thereby maintaining the high redox potential necessary for an electron carrier.

Acidithiobacillus↗
PubMed2003

Isolation and characterization of phenol-catabolizing bacteria from a coking plant.

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.

Aerobiosis↗
PubMed2003

Effect of power-frequency magnetic fields on genome-scale gene expression in Saccharomyces cerevisiae.

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (40 degrees C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

Cell Cycle↗
PubMed2002

Numerical simulation for electrochemical cultivation of iron oxidizing bacteria.

A numerical simulation model was constructed for electrochemical cultivation of iron oxidizing bacterium, Thiobacillus ferrooxidans, based on Monod's dual limitation equation. In this model, two limiting factors were examined, low supply of Fe(II) ion and dissolved oxygen, from empirical viewpoints. The simulation model was constructed taking into consideration the energy balance based on the amount of the electronic flow from the electrode to bacteria via an iron ion, and then to oxygen. The model consisted of a logarithmic bacterial growth phase during the first three days, followed by a plateau and growth limitation thereafter. The predicted results were in agreement with the actual growth under electrochemical cultivation. It was predicted the growth limiting factor would be changed from insufficient supply of Fe(II) ions to that of oxygen by decreasing the value of oxygen transfer constant K, which correlated with the aeration rate. The optimum aeration rate was determined for the ideal electrochemical cultivation. The algorithm described here can be used in any electrochemical cultivation by modifying the parameters for each system.

Bioreactors↗
PubMed2002

Photosynthetic productivity of conical helical tubular photobioreactor incorporating Chlorella sorokiniana under field conditions.

The photosynthetic performance of a conical, helical tubular photobioreactor (HTP) incorporating Chlorella sorokiniana was investigated under conditions of high temperature and light intensity during midsummer in an outdoor environment. Although the culture medium temperature exceeded 40 degrees C for approximately 5 h each day, peaking at 47.5 degrees C under sunny conditions, a photosynthetic productivity of 30.0 g x m(-2) (installation area) x day(-1) and a photosynthetic efficiency of 8.66% [photosynthetically active radiation (PAR), 400-700 nm] were achieved. A maximum photosynthetic productivity of 33.2 g x m(-2) x day(-1) was achieved on a sunny day, when solar energy input was also maximal (11.5 MJ x m(-2) x day(-1) [PAR]). On the other hand, a maximum photosynthetic efficiency of 9.54% was obtained on a day that was rainy in the morning and cloudy in the afternoon, and there was relatively little solar energy input. The average daily photosynthetic efficiency over the two culture periods (August 4 to 7 and August 10 to 13, 1999) was 7.25%. Thus, a high level of photosynthetic performance was achieved in the conical HTP incorporating Chlorella sorokiniana despite the fact that culture medium temperature was not controlled. The use of Chlorella sorokiniana in the conical HTP should be a good choice to produce microalgal biomass during the summer under field conditions.

Biomass↗
PubMed2002

Electrochemical regeneration of Fe(III) to support growth on anaerobic iron respiration.

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H(2) and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.

Anaerobiosis↗
PubMed2002

Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H(2)/O(2) under aerobic conditions but also on H(2)/Fe(3+), H(2)/S(0), or S(0)/Fe(3+) under anaerobic conditions. Anaerobic respiration using Fe(3+) or S(0) as an electron acceptor and H(2) or S(0) as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe(3+) induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe(3+). Respiration based on reduction of Fe(3+) coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.

Anaerobiosis↗