Biomedical subjects

Takeshi Kobayashi

Publications and source records attributed to Takeshi Kobayashi.

At least 19 recordsLinked to original sources

PubMed2026

A rotavirus vaccine candidate attenuated by codon deoptimization protects neonatal mice against wild-type virus infection.

Rotavirus infection is a leading cause of acute viral gastroenteritis and diarrhea in infants and young children. Owing to the limited development of effective antiviral therapies, vaccination has become the primary and most efficient strategy to reduce rotavirus-associated morbidity and mortality. Compared with classical virus attenuation strategies, reverse genetics approaches such as codon deoptimization are safer, more time-saving, more cost-effective, and more controllable. The present study describes the development of an oral live-attenuated rotavirus vaccine candidate using codon deoptimization. Based on a simian rotavirus SA11 strain, eight gene segments, encoding the structural proteins VP1, VP2, VP3, and VP6, and the non-structural proteins NSP2, NSP3, NSP4, and NSP5, were subjected to codon deoptimization. Attenuated rotavirus by multi-segment codon deoptimization (MS8cd) exhibited markedly attenuated replication both in vitro and in vivo, attributable to reduced protein production independent of mRNA stability. Despite the attenuation, MS8cd elicited robust systemic and mucosal antibody responses which were sufficient to protect neonatal mice against challenge with wild-type rotavirus in a maternal immunization model. To alter the immunogenicity, MS8cd was manipulated to encapsidate outer capsid proteins of several prevalent human rotaviruses. These reassortants exhibited altered antigenic and immunogenic properties associated with the differing genotypes of the outer capsid proteins. In conclusion, this study describes the generation of promising rotavirus vaccine candidates attenuated by codon deoptimization. They are capable of eliciting genotype-specific and broad-spectrum protective immunity against circulating strains of rotavirus. This represents a rapid-response platform for the development of novel vaccines against emerging variants.

Animals↗

PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗

PubMed2004

Development of agmatine sensor using the combination of putrescine oxidase and agmatinase for squid freshness.

Agmatine (Agm) is an indicator of squid freshness. The Agm sensor was developed using flow injection analysis (FIA) that consisted of the putrescine oxidase (PuOx) reactor, the agmatinase (AUH)-PuOx reactor and two oxygen electrodes. In the proposed sensor, the first step is that coexisting cadaverine (Cad) and putrescine (Put) are removed by passing through the PuOx reactor and the initial decomposition is determined by the amount of oxygen consumed, simultaneously. The second step is that the amount of Agm is determined by the amount of oxygen consumed in the AUH-PuOx reactor. The optimum conditions for the use of the Agm sensor were as follows: 50 mM HEPES containing MnSO4 at a final concentration of 5 mM, pH 8.0, flow rate of 0.6 mL min(-1) and injection volume of 50 microL. A single assay could be completed in approximately 3 min. A linear relationship was obtained between the output and the Agm concentration in the range of 0.01-1 mM Agm with a correlation coefficient of 0.999. The detection limit was 0.005 mM. The relative standard deviations (RSDs) were 3.14 and 1.19% (n = 20) for 0.1 and 0.3 mM Agm, respectively. The extracts of squid were injected into the proposed sensor and the results were compared with those obtained using the conventional high-performance liquid chromatography (HPLC) method. A correlation was observed between the results obtained by the proposed sensor and those obtained by the conventional method. The determination of squid freshness is one of the good uses of the proposed Agm sensor.

Agmatine↗

PubMed2004

Artificial neural network approach for selection of susceptible single nucleotide polymorphisms and construction of prediction model on childhood allergic asthma.

BACKGROUND: Screening of various gene markers such as single nucleotide polymorphism (SNP) and correlation between these markers and development of multifactorial disease have previously been studied. Here, we propose a susceptible marker-selectable artificial neural network (ANN) for predicting development of allergic disease. RESULTS: To predict development of childhood allergic asthma (CAA) and select susceptible SNPs, we used an ANN with a parameter decreasing method (PDM) to analyze 25 SNPs of 17 genes in 344 Japanese people, and select 10 susceptible SNPs of CAA. The accuracy of the ANN model with 10 SNPs was 97.7% for learning data and 74.4% for evaluation data. Important combinations were determined by effective combination value (ECV) defined in the present paper. Effective 2-SNP or 3-SNP combinations were found to be concentrated among the 10 selected SNPs. CONCLUSION: ANN can reliably select SNP combinations that are associated with CAA. Thus, the ANN can be used to characterize development of complex diseases caused by multiple factors. This is the first report of automatic selection of SNPs related to development of multifactorial disease from SNP data of more than 300 patients.

Alleles↗

PubMed2004

Magnetite nanoparticle-loaded anti-HER2 immunoliposomes for combination of antibody therapy with hyperthermia.

Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexpressing cancer. Previously, we produced hyperthermia using magnetite nanoparticles that generate heat in an alternating magnetic field. In the present study, we constructed anti-HER2 immunoliposomes containing magnetite nanoparticles, which act as tumor-targeting vehicles, combining anti-HER2 antibody therapy with hyperthermia. The magnetite nanoparticle-loaded anti-HER2 immunoliposomes exerted HER2-mediated antiproliferative effects on SKBr3 breast cancer cells in vitro. Moreover, 60% of magnetite nanoparticles were incorporated into SKBr3, and the cells were then heated at 42.5 degrees C under an alternating magnetic field, resulting in strong cytotoxic effects. These results suggest that this novel therapeutic tool is applicable to treatment of HER2-overexpressing cancer.

Antibodies, Monoclonal↗

PubMed2004

Construction of robust prognostic predictors by using projective adaptive resonance theory as a gene filtering method.

MOTIVATION: For establishing prognostic predictors of various diseases using DNA microarray analysis technology, it is desired to find selectively significant genes for constructing the prognostic model and it is also necessary to eliminate non-specific genes or genes with error before constructing the model. RESULTS: We applied projective adaptive resonance theory (PART) to gene screening for DNA microarray data. Genes selected by PART were subjected to our FNN-SWEEP modeling method for the construction of a cancer class prediction model. The model performance was evaluated through comparison with a conventional screening signal-to-noise (S2N) method or nearest shrunken centroids (NSC) method. The FNN-SWEEP predictor with PART screening could discriminate classes of acute leukemia in blinded data with 97.1% accuracy and classes of lung cancer with 90.0% accuracy, while the predictor with S2N was only 85.3 and 70.0% or the predictor with NSC was 88.2 and 90.0%, respectively. The results have proven that PART was superior for gene screening. AVAILABILITY: The software is available upon request from the authors. CONTACT: honda@nubio.nagoya-u.ac.jp

Algorithms↗

PubMed2004

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa.

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.

Biosensing Techniques↗

PubMed2004

Analysis of adsorption equilibrium of volatile chlorinated organic compounds to dry soil.

Adsorption is one of the main mechanisms of soil contamination by hazardous volatile chlorinated organic compounds. The adsorption equilibriums of six volatile organic chlorinated compounds to three dry soils were investigated using batch adsorption experiments. The adsorption equilibriums for the dry soils could be expressed by the Dubinin-Astakhov equation. The equation's parameters were analyzed with the characteristic values of the soils and compounds. No correlation between the values of the affinity coefficients, beta, and the molecular volume, Mv, was found. W(0) could be expressed by the functions of a pore volume of less than 10 nm, V(<10 nm), or the specific surface area, S. The adsorbed amount could be estimated using equations relating E(0), V(<10 nm) (or S), and beta. The predicted amounts agreed well with the measured data.

Adsorption↗

PubMed2004

Single-molecule imaging analysis of Ras activation in living cells.

A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.

Boron Compounds↗

PubMed2004

Epitope mapping of the melanosomal matrix protein gp100 (PMEL17): rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment.

Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100. We now report that gp100 is processed and sorted in a manner distinct from other melanosomal proteins (such as tyrosinase, Tyrp1 and Dct) and is predominantly delivered directly to immature melanosomes following its rapid processing in the endoplasmic reticulum and cis-Golgi. Following its arrival, gp100 is cleaved at the amino and at the carboxyl termini in a series of specific steps that result in the reorganization of immature melanosomes to the fibrillar mature melanosomes. Once this structural reorganization occurs, melanogenic enzymes begin to be targeted to the melanosomes, which are then competent to synthesize melanin pigment.

Antibodies, Monoclonal↗

PubMed2004

Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma.

BACKGROUND: We have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. METHOD: MCLs were injected into the osteosarcoma and then subjected to an AMF. RESULTS: The tumor was heated at over 42 degrees C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. CONCLUSION: These results suggest that this treatment is effective for osteosarcoma.

Journal Article↗

PubMed2004

Single-channel activity of L-type Ca2+ channels reconstituted with the beta2c subunit cloned from the rat heart.

We recently cloned the beta(2c) subunit of the L-type Ca(2+) channel as a functional type of beta subunit from the rat heart. In order to clarify the contribution of the beta(2c) subunit to native Ca(2+) channel function, we investigated the single-channel properties of Ca(2+) channels reconstituted with beta(2a) or beta(2c) subunits and compared them with the properties of native channels. In contrast to the Ca(2+) channel with beta(2a) subunit, long-lasting closings were dominant in the Ca(2+) channel with beta(2c) subunit and the native channel. The ensemble-averaged current of the cells with beta(2c) subunits was comparable to that of the native cardiomyocytes. Many high P(o) sweeps (mode 2) were observed in the cells with beta(2a) subunits, while only a few high P(o) sweeps were observed in the cells with beta(2c) subunits and the native cells. These findings suggest that the beta(2c) subunit is one of the functional beta subunits in the rat heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2004

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array.

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.

Amino Acid Sequence↗

PubMed2004

Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M.

Little is known about the mechanisms involved in the dysfunction of melanocytes in vitiligo epidermis. It is hypothesized that some cytokine/receptor interactions may be affected, resulting in dysfunction and/or loss of melanocytes. This study has compared the expression of endothelin (ET)-1, the ET-1 receptor (ET(B)R), granulocyte macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), the SCF receptor (KIT protein), tyrosinase, and S100 alpha between lesional and non-lesional vitiligo epidermis. Analysis by reverse transcription-polymerase chain reaction (RT-PCR) and by western blotting for ET-1 and SCF unexpectedly demonstrated up-regulated expression of these cytokines in lesional vitiligo epidermis. Immunohistochemistry with antibodies to melanocyte markers revealed that at the edge of the lesional epidermis, melanocytes remain and express tyrosinase, S100 alpha and ET(B)R, but not KIT protein or melanocyte-specific microphthalmia-associated transcription factor (MITF-M). Quantitation of the staining revealed a slight or moderate decrease in the number of S100 alpha, tyrosinase, and ET(B)R-positive cells at the edge of the lesional epidermis. In contrast, the number of cells expressing KIT protein was markedly decreased at the edge of the lesional epidermis compared with the non-lesional epidermis. At the centre of the lesional epidermis, there was complete loss of melanocytes expressing KIT protein, S100 alpha, ET(B)R, and/or tyrosinase. Western blotting revealed down-regulated expression of c-kit and MITF-M proteins at the edge of the lesional epidermis in vitiligo. These findings suggest that reduction in the expression of KIT protein by melanocytes and its downstream effectors, including MITF-M, may be associated with the dysfunction and/or loss of melanocytes in vitiligo epidermis.

Adult↗

PubMed2004

Industrial application of fuzzy control in bioprocesses.

In a bioprocess, for example a fermentation process, many biological reactions are always working in intracellular space and the control of such a process is very complicated. Bioprocesses have therefore been controlled by the judgment of the experts who are the skilled operators and have much experience in the control of such processes. Such experience is normally described in terms of linguistic IF-THEN rules. Fuzzy inference is a powerful tool for incorporating linguistic rules into computer control of such processes. Fuzzy control is divided into two types--direct fuzzy control of process variables, for example sugar feed rate and fermentation temperature, and indirect control via phase recognition. In bioprocess control the experts decide the value of controllable process variables such as sugar feed rate or temperature as output data from several state variables as input data. Fuzzy control is regarded as a computational algorithm in which the causal relationship between input and output data are incorporated. In Japan fuzzy control has already been applied to practical industrial processes such as production of pravastatin precursor and vitamin B2 and to the Japanese sake mashing process; these examples are reviewed. In addition, an advanced control tool developed from a study on fuzzy control, fuzzy neural networks (FNN), are introduced. FNN can involve complicated causality between input and output data in a network model. FNN have been proven to be applicable to a research in biomedicine, for example modeling of the complicated causality between electroencephalogram or gene expression profiling data and prognostic prediction. Successful results on this research will be also explained.

Algorithms↗

PubMed2004

Large-scale micropropagation system of plant cells.

Plant micropropagation is an efficient method of propagating disease-free, genetically uniform and massive amounts of plants in vitro. The scale-up of the whole process for plant micropropagation should be established by an economically feasible technology for large-scale production of them in appropriate bioreactors. It is necessary to design suitable bioreactor configuration which can provide adequate mixing and mass transfer while minimizing the intensity of shear stress and hydrodynamic pressure. Automatic selection of embryogenic calli and regenerated plantlets using image analysis system should be associated with the system. The aim of this chapter is to identify the problems related to large-scale plant micropropagation via somatic embryogenesis, and to summarize the micropropagation technology and computer-aided image analysis. Viscous additive supplemented culture, which is including the successful results obtained by us for callus regeneration, is also introduced.

Alginates↗

PubMed2004

Pancreatic metastasis of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a relatively rare skin tumor that is considered to have intermediate malignancy; it demonstrates frequent local recurrence, but systemic metastasis is rare. We report a 49-year-old woman with pancreatic metastasis of DFSP who underwent total pancreatectomy with partial resection of the portal vein. Except for our patient, only two other cases of pancreatic metastasis of DFSP have been reported in the literature, to our knowledge. Radical resection may be considered for pancreatic metastasis of DFSP when there are no other metastatic lesions.

Abdominal Neoplasms↗

PubMed2004

Long-term pain relief effects in four patients undergoing percutaneous vertebroplasty for metastatic vertebral tumor.

We reviewed long-term pain relief in four patients undergoing percutaneous vertebroplasty (PVP) for lumbar or back pain due to metastatic vertebral tumors. The patients received anesthesiological palliative care with analgesics until their death after PVP. Pain intensity, the presence or absence of recurrence of pain, changes in dosage of analgesics given before and after PVP, and complications associated with PVP were evaluated. A numerical rating scale (NRS) from 0 (no pain) to 10 (worst pain imaginable) was used to measure pain. The patients were three men and one woman (mean age, 58 years). PVP was performed in five vertebrae (one thoracic and four lumbar). The NRS scores on moving before PVP were 10, 8, 10, and 10. After PVP, NRS decreased to 0, 3, 5, and 0, respectively, within 72 h. No recurrence of pain in the treated area occurred until death in any of these patients. The dosages of analgesics given were decreased in two cases, but no changes were made in the other two cases. There were no complications associated with PVP. Percutaneous vertebro-plasty is a safe and effective treatment for long-time pain relief in patients with metastatic vertebral tumors.

Aged↗