Biomedical subjects

Shigeo Katoh

Publications and source records attributed to Shigeo Katoh.

16 recordsLinked to original sources

PubMed2006

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface.

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.

Affinity Labels↗

PubMed2006

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column.

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.

Base Pair Mismatch↗

PubMed2006

Effects of methanol feeding methods on chimeric alpha-amylase expression in continuous culture of Pichia pastoris.

The effects of two types of methanol feeding methods in a continuous culture of Pichia system on the cell growth and recombinant protein expression were studied using chimeric alpha-amylase as a model protein. With the feeding of methanol by a DO-stat method, the alpha-amylase concentration in the fermentation broth increased with decreasing dilution rate and reached 173 mg/l at a dilution rate of 0.013 h(-1), at which the maximum volumetric productivity of alpha-amylase was obtained. Although almost the same productivity was attained at 0.04 h(-1) with continuous methanol feeding, the alpha-amylase concentration was one third that compared with feeding by the DO-stat method, that is, 55 mg/l. Furthermore, at this dilution rate, the medium volume needed per unit time was three times that required when DO-stat was used. Therefore, continuous culture with methanol feeding by the DO-stat method may be a promising method for the production of recombinant proteins on an industrial scale by Pichia pastoris.

Bioreactors↗

PubMed2006

Degradation of cyanuric acid in soil by Pseudomonas sp. NRRL B-12227 using bioremediation with self-immobilization system.

The rates of degradation of cyanuric acid, a key intermediate in a metabolic pathway of s-triazine herbicides, were measured for Pseudomonas sp. NRRL B-12227. The rate of degradation was affected by the rate of cyanuric acid transport through cell membranes and the activity of cyanuric acid amidohydrolase inside the cells. At low concentrations of cyanuric acid, the acclimation of cells to cyanuric acid and/or added nutrients effectively enhanced the degradation rate. The strain was also applied to bioremediation using a Bioremediation with Self-Immobilization System (BSIS), in which Pseudomonas sp. NRRL B-12227 cells were co-immobilized with Bacillus subtilis, the latter of which secretes a viscous polymer, in a shallow layer of soil packed in a column. More than 70% of the Pseudomonas sp. NRRL B-12227 cells were co-immobilized with the B. subtilis in a 7.5 cm layer of the packed soil by self-aggregation. More than 60% of the 1 mM cyanuric acid supplied to the packed soil was degraded in this layer during a 72 h period.

Biodegradation, Environmental↗

PubMed2006

Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis.

To conserve energy in the production of astaxanthin by the green alga Haematococcus pluvialis, we utilized intermittent flashing light from blue light emitting diodes (LEDs) and investigated the effects of the incident light intensity (2-12 micromol m(-2) s(-1)), duty cycle (17-67%) and frequency (25-200 Hz) of flashing on the cell growth and astaxanthin production. In the above ranges, the final astaxanthin concentration under illumination by flashing light was significantly higher than that obtained under illumination with continuous light at the same incident intensity. For example, flashing light at an incident intensity of 8 micromol m(-2) s(-1) gave the same final astaxanthin concentration that was obtained under continuous light illumination at 12 micromol m(-2) s(-1), thus reducing energy consumption by 1/3. We therefore conclude that flashing light from blue LEDs is a promising illumination method for indoor algal cultivation using photobioreactors.

Bioreactors↗

PubMed2006

Effective utilization of transmitted light for astaxanthin production by Haematococcus pluvialis.

A new cultivation method, in which physiological responses of Haematococcus pluvialis to different intensities and wavelengths of illuminating light are utilized, is proposed. In this method, light transmitted through a cultivation vessel illuminated from the opposite side was utilized for the early-phase cultivation of H. pluvialis in another inoculated vessel that was located behind the cultivation vessel, to save time required for the growth of cells. After harvesting cells from the front vessel, the vessel that was originally behind was shifted to the position of the front vessel. The abrupt increase in light intensity caused by shift of the position induced the effective accumulation of astaxanthin. These procedures for inoculation, shift of vessels and harvest were repeated using two vessels arranged in series along the light path. After four repeated cycles of cultivation (840 h from the start of cultivation), using 2.5 cm thick vessels, astaxanthin production per unit volume and the astaxanthin content were higher than obtained in a batch cultivation with two 2.5 cm vessels.

Bioreactors↗

PubMed2005

Application of protein-coupled liposomes to effective affinity screening from phage library.

For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of dipalmitoylphosphatidylcholine (DPPC): dicetylphosphate (DCP) = 10: 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After four rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15% of the selected phages displayed homological peptides in the conventional biopanning. The octapeptide selected by AB-MLVs against the anti-peptide antibody showed comparable binding affinity, which were determined by the competitive ELISA and an immunoaffinity chromatography, to that of the peptide antigen. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.

Affinity Labels↗

PubMed2005

Fed-batch culture under illumination with blue light emitting diodes (LEDs) for astaxanthin production by Haematococcus pluvialis.

To increase the cell concentration and the accumulation of astaxanthin, the effects of the fed-batch addition of 10-fold-concentrated medium to supply nutrients, as well as illumination with blue light emitting diodes (LEDs), on cell growth and accumulation of astaxanthin were studied for the cultivation of Haematococcus pluvialis. Using the fed-batch addition method, the cell concentration increased above 1 mg-dry cell/cm3, and under illumination with blue LEDs, the astaxanthin concentration reached approximately 70 microg/cm3. This method was much simpler to operate than the medium replacement method in operation and enabled us to attain a higher total yield of astaxanthin.

Biotechnology↗

PubMed2005

Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus.

Using a semicontinuous culture method, in which operational parameters such as cell concentration and light intensity distribution were maintained almost constant, instability of the specific growth rate of Rhodobacter capsulatus B-100, a purple bacterium, was observed to be similar to that of R. capsulatus ST-410 when cultivated under high ratios of light intensity on the illuminated side to that of the transmitted light. Such instability was not observed in the cultivation of Chlorella vulgaris, a eukaryotic green alga, even at higher cell concentrations. Under the same conditions, the increase in only the ferrous concentration from 43 microM, the concentration in the original RCV medium, to 172 microM sustained a stable growth, whereas Fe(2+) was slightly consumed during the cultivation. Supplemental illumination with a fluorescent lamp on the transmitted side of a flat plate photobioreactor sustained a moderate level of stable growth, while a halogen lamp slightly affected the growth stability. Our results showed that an increase in Fe(2+) concentration or supplemental illumination improves the growth stability of R. capsulatus.

Bioreactors↗

PubMed2004

Characteristics of neutralization of acids by newly isolated fungal cells.

Soil microorganisms play an important role in maintaining soil pH at levels suitable for other soil organisms. To clarify the biological neutralization mechanism in soil, we isolated soil microorganisms showing a high ability to neutralize acids and studied their characteristics. From our taxonomic study, three isolated strains were identified as filamentous fungi, namely Mucor sp., Aspergillus fumigatus, and Aureobasidium pullulans. These strains could secrete basic materials, such as ammonia, for neutralization, grow in the medium at pH 4.0 and increase the pH of the medium to approximately 8.0. These microbial cells could neutralize not only nitric acid but also sulfuric and hydrochloric acids. The strains could also grow by utilizing nitric acid as a sole nitrogen source. In the soil containing these organisms, the pH was maintained in the neutral range by the buffering action of basic materials that they secrete. These results suggest that these fungal cells are useful for protecting the soil from acidification by acid rain.

Journal Article↗

PubMed2004

Characteristics of microblotting assay using immunoliposomes.

To clarify the applicability of a liposome immunoblotting assay to microassay systems, the effects of the sample volume blotted on polyvinylidiene fluoride (PVDF) membrane on the sensitivity and signal intensity were studied and compared with those in a conventional immunoblotting assay utilizing enzyme-labeled antibody. In the liposome immunoblotting assay, signal intensities per unit area (signal density) were almost the same or increased slightly with decreasing blotted sample volume (from 20 to 2 microl) at the same concentration of analyte (human IgM) adsorbed on PVDF membrane. A substrate, 4-chloro-1-naphtol, for color development formed colored precipitates inside the liposomes. Thus, the colored signal was detected without loss of intensity. On the other hand, in the conventional immunoblotting assay, signal densities decreased with decreasing sample volume at the same concentration of analyte. This is caused by partial losses of the colored signal into the substrate solution by diffusion, because the fraction of loss increases with decreasing sample volume. These results show that the liposome immunoblotting assay is a promising method for the detection of analytes blotted in small areas, because its sensitivity is higher than that of the conventional blotting assay.

Journal Article↗

PubMed2004

Effects of nutrient supply methods and illumination with blue light emitting diodes (LEDs) on astaxanthin production by Haematococcus pluvialis.

In order to increase the cell concentration and the accumulation of astaxanthin, the effects of nutrient concentration, pH, illumination and methods of supplying nutrients were studied for the cultivation of Haematococcus pluvialis. The replacement of media to avoid the deficiency of nutrients increased the cell concentration above 1 mg-dry cell cm(-3) without induction of astaxanthin accumulation. Illumination with blue light emitting diode lamps and nutrient starvation induced accumulation of astaxanthin, and the interactive effects of these two increased the astaxanthin concentration to 76 mug cm(-3).

Journal Article↗

PubMed2004

High performance in protease refolding by application of a continuous system using immobilized inhibitor.

In the reoxidative refolding of Streptomyces griseus trypsin, which is a serine protease having three S-S bonds per molecule, a synthetic inhibitor immobilized on agarose beads was applied in order to avoid the digestion of non-renatured protease molecules by renatured ones. A semi-continuous refolding system was fabricated by packing such inhibitor-immobilized gels in a glass column and the optimal operating conditions were investigated. Taking account of the effective conditions surveyed in the system, a continuous refolding system was constructed and operated to achieve higher performance. By application of the continuous system, a marked increase in the recovery rate as well as high recovered activity could be accomplished, i.e., the recovery rate obtained was ca. 40 times higher than that in the semi-continuous system. This system was also revealed to be substantially advantageous since it includes not only effective refolding but also separation, purification and enrichment processes in one operation.

Journal Article↗

PubMed2002

Liposome immunoblotting assay using a substrate-forming precipitate inside immunoliposomes.

A new immunoblotting assay which uses antibody-coupled liposomes containing horseradish peroxidase is proposed. A substrate 4-chloro-1-naphthol permeated through the phospholipid membrane of the antibody-coupled liposomes and formed a colored product precipitating inside the liposomes. The precipitates accumulated in the liposomes and could be detected at the positions where the liposomes coupled with a target in blotted samples. Combination of liposomes with average diameter of 350 nm and a PVDF membrane with a pore size of 450 nm, 0.02 ng of IgM was detected, while the conventional immunoblotting using antibody-HRP conjugates detected 2 ng of IgM. The sensitivity increased about two orders of magnitude by the liposome immunoblotting assay. This liposome immunoblotting assay gives a simple detection method of proteins with a high sensitivity, as well as a high sensitivity Western blotting assay.

Blotting, Western↗

PubMedDate not supplied

Effects of light intensity distribution on growth of Rhodobacter capsulatus.

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.

Adaptation, Physiological↗

PubMedDate not supplied

Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins.

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.

Absorption↗