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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↗
PubMed2007

Electrophysiologic changes in dorsal root ganglion neurons and behavioral changes in a lumbar radiculopathy model.

STUDY DESIGN: The DRG neuron was electrophysiologically investigated using a rat model with constriction of the proximal site of the DRG. OBJECTIVES: To investigate the pathomechanisms of lumbar radiculopathy, we established a rat model with constriction of the proximal site of the DRG. And to characterize the DRG neurons in the rat model of lumbar radiculopathy, the physiologic properties regarding action potential, Na, and K current of the DRG neurons were analyzed through the use of patch clamp recordings. SUMMARY OF BACKGROUND DATA: In lumbar root constriction models, properties of secondary afferent neurons in the dorsal horn have been investigated. However, the electrical properties of DRG neuron have not been well investigated. METHODS: To compare the excitability of DRG neurons between root constriction models and sham, we examined the threshold current, action potential (AP) threshold, resting membrane potential (RMP), afterhyperpolarization (AHP), action potential duration 50 (APD50), action potential amplitude, maximum rise time of AP, and pattern of discharges evoked by depolarizing current. We also examined the peak Na current and steady-state Na and K currents with the voltage clamp technique. RESULTS: The rats in the root constriction group demonstrated mechanical allodynia and thermal hyperalgesia. In measurement of the action potential, lower threshold current, more depolarized RMP, larger AHP, and prolonged APD50 were measured in the root constriction neurons compared with the sham group. The incidence of sustained burst was significantly higher in root constriction neurons. The Na current in root constriction neurons was markedly larger. There were no significant differences in K current density and voltage dependency. CONCLUSIONS: The constriction of lumbar root increased excitability and Na current amplitude of DRG neurons. These findings indicate that lumbar radicular pain may be associated with increased excitability of involved DRG neurons.

Action Potentials↗
PubMed2007

Distribution and diversity of tetracycline resistance genes encoding ribosomal protection proteins in Mekong river sediments in Vietnam.

We investigated the distribution and diversity of tetracycline resistance genes encoding ribosomal protection proteins (RPPs) in river and channel sediments of the Mekong Delta in Vietnam. The sediment samples were taken from nine sites in the Hau River in southern Vietnam and from 1 site in a channel in Can Tho City in May 2004 using an Ekman-Birge sediment surface sampler. The RPP genes were amplified using PCR with DNA templates obtained directly from the sediments. The tet(M), tet(S), and tet(W) genes were detected by PCR in most sediment samples. Denaturing gradient gel electrophoresis analysis of these genes and sequencing of the resulting bands showed that tet(S) and tet(W) had only one genotype each, but that tet(M) had at least two, which were tentatively called type 1 and type 2. Type 1 tet(M) was identical to the gene encoded in various plasmids and transposons of gram-positive and gram-negative bacteria, and type 2tet(M) was similar to the gene encoded in Tn1545 of Enterococcus faecalis (99% identity, 170 bp/171 bp). This study showed that various RPP genes were widely distributed in the river and channel sediments of the Mekong Delta.

Amino Acid Sequence↗
PubMed2006

Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids.

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗
PubMed2006

Particle-size effect of nanoscale platinum catalysts in oxygen reduction reaction: an electrochemical and 195Pt EC-NMR study.

Oxygen reduction reaction (ORR) measurements and (195)Pt electrochemical nuclear magnetic resonance (EC-NMR) spectroscopy were combined to study a series of carbon-supported platinum nanoparticle electrocatalysts (Pt/CB) with average diameters in the range of roughly 1-5 nm. ORR rate constants and H(2)O(2) yields evaluated from hydrodynamic voltammograms did not show any particle size dependency. The apparent activation energy of 37 kJ mol(-1), obtained for the ORR rate constant, was identical to that obtained for bulk platinum electrodes. Pt/CB catalysts on Nafion produced only 0.7-1% of H(2)O(2), confirming that the direct four-electron reduction of O(2) to H(2)O is the predominant reaction. NMR spectral features showed characteristic size dependence, and the line shapes were reproduced by using the layer-deconvolution model. Namely, the variations in the NMR spectra with particle size can be explained as due to the combined effect of the layer-by-layer variation of the s-type and d-type local density of states. However, the surface peak position of (195)Pt NMR spectra and the spin-lattice relaxation time of surface platinum atoms showed practically no change with the particle size variation. We conclude that there is a negligible difference in the surface electronic properties of these Pt/CB catalysts due to size variations and therefore, the ORR activities are not affected by the differences in the particle size.

Catalysis↗
PubMed2006

A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle.

The lymphoid tissue in the omentum, at the so-called milky spots, is known as an initial place for disseminated cancer cells to develop into solid tumors. In the present study, i.p. macrophages significantly took up oligomannose-coated liposomes (OMLs) that were injected into the peritoneal cavity, and then gradually accumulated in the omentum and the other lymphoid tissues within 24 hours of i.p. injection of OMLs. When 5-fluorouracil (5-FU) was encapsulated in the OMLs, >60% of administered 5-FU accumulated in the omentum. Treatment of macrophages at 39 degrees C for 30 minutes led to the release of 5-FU from the macrophages, suggesting that controlled release from macrophages could be achieved by mild hyperthermia. We encased magnetic nanoparticles, which are known to convert electromagnetic energy to heat in the OMLs to achieve in vivo hyperthermia at the site. Using this system in a mouse i.p. metastasis model, we successfully controlled tumor development by coadministration of OML-encased 5-FU and OML-encased magnetic nanoparticles, followed by treatment with an alternating magnetic field. No apparent reduction was seen in tumor growth with the administration of OML-encased magnetic nanoparticles or OML-encased 5-FU alone. Thus, we have established the use of i.p. macrophages as a novel drug delivery system for the control of cancer metastatic to milky spots.

Animals↗
PubMed2006

Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus).

In recent years, probiotics, especially lactic acid bacteria, have been used as dietary supplements to protect fish from various infections. Here, we examined the protective effects of Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Cumulative mortality was significantly lower in probiotic-supplemented fish than in control fish. In a histopathological survey, pyogranulomatous responses were observed at an earlier stage and to a greater extent in the probiotic-supplemented fish than in the control fish. Immunohistochemistry using an anti-E. tarda antibody revealed a larger number of positive signals in pyogranuloma-participating cells, indicating an enhanced phagocytic ability. Alternative complement activity was significantly higher in the probiotic groups than in the control. These results suggest that L. rhamnosus enhanced the alternative complement system of the fish, enabling phagocytic cell aggregation, increasing phagocytic activity and subsequently protecting the fish from acute septicemic death by E. tarda infection. Prevention of thymic necrosis by the probiotic supplement seems to minimize immunosuppression and to initiate an immune response against edwardsiellosis.

Animals↗
PubMed2006

Evaluating the fish safety level of river water and wastewater with a larval medaka assay.

A larval medaka (Oryzias latipes) assay was proposed to evaluate the fish safety level of river waters and wastewaters. Organic toxicants were 10-100 times concentrated with adsorption cartridges from 4 l of river water or 1-10 times concentrated from 400 ml of wastewater. Toxicity of these concentrated solutions was determined by exposing 48-72 h post-hatch age larvae for 48 h. The method effectively revealed a variation of the median lethal concentration ratio (LCR50) from 13 to >100 in 125 river water samples, and from <1 to >10 in five typical wastewater samples. Ayase River, which takes water mostly from agricultural or household discharge, showed significantly (P<0.001) lower LCR50 than Sagami River that takes natural water as the source. Safety Levels in both Sagami River and Ayase River were influenced by the irrigation activity, LCR50 at some sites showing a seasonal-specific decrease in winter. Pollution from pulp and paper industries contributed to the low LCR50 in several tributaries of Ayase River. Required little manpower in sampling, pretreatment and testing, the proposed larval medaka assay was proved as an efficient tool for screening those high risk sites for priority management.

Animals↗
PubMed2006

Degradation of tributyltin in microcosm using Mekong River sediment.

The degradation of tributyltin (TBT) and changes of bacterial number and community structures were investigated in microcosms using the sediment collected from the Mekong River, Vietnam. Concentrations of TBT in sediments were less than 0.62 ng/g (dry wt), lower than those reported from other areas. TBT-resistant bacteria were found in the three sampling sites, and the occurrence rates were 11-16% out of the total viable count. In this microcosm experiment, initial concentration of TBT [1.0-1.4 microg/g (dry wt)] decreased to 0.6 microg/g (dry wt) during 150 days, whereas that in the control microcosm with autoclaved sediment did not change, indicating that Mekong River sediment contains high TBT-degrading activity by microorganisms. The occurrence of TBT-resistant bacteria and the bacterial community structures monitored by denaturing gradient gel electrophoresis were almost the same between test and control groups, indicating that the addition of TBT had little influence on microbial community structure. Mekong River sediment seems to have a stable microbial community against TBT pollution.

Bacteria↗
PubMed2006

Complete regression of experimental prostate cancer in nude mice by repeated hyperthermia using magnetite cationic liposomes and a newly developed solenoid containing a ferrite core.

BACKGROUND: Magnetite cationic liposomes (MCLs) can be used to induce hyperthermia because they generate heat in an alternating magnetic field (AMF). This study aimed at developing more practical method for MCL hyperthermia examining the effect of MCL-induced hyperthermia on human prostate cancer in vivo. MATERIALS AND METHODS: A newly developed AMF generator incorporating a solenoid with a ferrite core (FC) was used. Human prostate cancer cells (PC-3 and LNCap) were injected subcutaneously into nude mice. MCLs were injected into tumor nodule and the mice were exposed into AMF three times at 24-hr intervals (repeated hyperthermia; RH) until complete tumor regression was observed. RESULTS: Irradiation with an AMF generated by newly developed device can adequately increase the temperature of tumor tissue. Frequent RH resulted in complete tumor regression in all nude mice. CONCLUSION: RH using MCLs may be a promising new therapy for hormone-refractory human prostate cancer in the future.

Animals↗
PubMed2006

Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal.

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses.

Active Transport, Cell Nucleus↗
PubMed2006

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein-protein interactions.

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.

Animals↗
PubMed2006

High-throughput screening of cell death inducible short peptides from TNF-related apoptosis-inducing ligand sequence.

Therapeutic peptides and small molecules, rationally designed to trigger cell death have attracted strong attention. Cell death inducible peptides were screened from amino acid sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using Fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of TRAIL scan was prepared and cell viability assay was directly performed on peptide disk with Jurkat cells. Six peptide sequences that could induce cell death were found. Peptide sequence with RNSCWSKD (TRAIL(227-234)) that exist in the zinc-binding site revealed high cell death inducible activity. Apoptotic cell death was observed when cells were treated with soluble synthesized peptide.

Amino Acid Sequence↗
PubMed2006

Computerized methods for determining respiratory phase on dynamic chest radiographs obtained by a dynamic flat-panel detector (FPD) system.

Chest radiography using a dynamic flat-panel detector with a large field of view can provide sequential chest radiographs during respiration. These images provide information regarding respiratory kinetics, which is effective for diagnosis of pulmonary diseases. For valid analysis of respiratory kinetics in diagnosis of pulmonary diseases, it is crucial to determine the association between the kinetics and respiratory phase. We developed four methods to determine the respiratory phase based on image information associated with respiration and compared the results in dynamic chest radiographs of 37 subjects. Here, the properties of each method and future tasks are discussed. The method based on the change in size of the lung gave the most stable results, and that based on the change in distance from the lung apex to the diaphragm was the most promising method for determining the respiratory phase.

Humans↗
PubMed2006

Pathologic and biological assessment of lung tumors showing ground-glass opacity.

BACKGROUND: We evaluated the pathologic and biological aspects of lung tumors 3.0 cm or less in diameter with the appearance of ground-glass opacity (GGO). METHODS: Of 988 patients with non-small cell lung cancer who underwent operations at our institute between January 1994 and December 2004, 87 resected lung tumor specimens that showed GGO appearance on helical computed tomography were obtained from 81 patients. Forty-four lesions were pure GGO with no solid component in the tumor and 43 lesions were mixed GGO consisting of areas of attenuation with a solid component. Together with histological features, MIB1 and nm23 expression within tumors were examined immunohistochemically. RESULTS: The mean tumor size in the pure GGO group was significantly smaller than that in the mixed GGO group. The composition of pathologic subtypes and biological characteristics were clearly different between the two groups. Although atypical adenomatous hyperplasia and localized bronchioloalveolar cell carcinoma of Noguchi's A and B were the predominant pathologic subtypes and nm23 negativity was rare in the pure GGO group, a high score for expression of MIB1 was often found in pure GGO tumors even though the tumors were less than 10 mm in diameter. CONCLUSIONS: If the tumor is 2 cm or less in diameter, the ability of invasion and metastasis seems to be low in pure GGO tumors. However, the proliferation ability of these tumors suggests the necessity of a careful follow-up schedule if the tumor is greater than 5 mm in diameter. For mixed GGO tumors, surgical resection instead of observation is justified.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Evaluation of pulmonary function using breathing chest radiography with a dynamic flat panel detector: primary results in pulmonary diseases.

OBJECTIVES: Dynamic flat panel detectors (FPD) permit acquisition of distortion-free radiographs with a large field of view and high image quality. The present study was performed to evaluate pulmonary function using breathing chest radiography with a dynamic FPD. We report primary results of a clinical study and computer algorithm for quantifying and visualizing relative local pulmonary airflow. MATERIALS AND METHODS: Dynamic chest radiographs of 18 subjects (1 emphysema, 2 asthma, 4 interstitial pneumonia, 1 pulmonary nodule, and 10 normal controls) were obtained during respiration using an FPD system. We measured respiratory changes in distance from the lung apex to the diaphragm (DLD) and pixel values in each lung area. Subsequently, the interframe differences (D-frame) and difference values between maximum inspiratory and expiratory phases (D-max) were calculated. D-max in each lung represents relative vital capacity (VC) and regional D-frames represent pulmonary airflow in each local area. D-frames were superimposed on dynamic chest radiographs in the form of color display (fusion images). The results obtained using our methods were compared with findings on computed tomography (CT) images and pulmonary functional test (PFT), which were examined before inclusion in the study. RESULTS: In normal subjects, the D-frames were distributed symmetrically in both lungs throughout all respiratory phases. However, subjects with pulmonary diseases showed D-frame distribution patterns that differed from the normal pattern. In subjects with air trapping, there were some areas with D-frames near zero indicated as colorless areas on fusion images. These areas also corresponded to the areas showing air trapping on computed tomography images. In asthma, obstructive abnormality was indicated by areas continuously showing D-frame near zero in the upper lung. Patients with interstitial pneumonia commonly showed fusion images with an uneven color distribution accompanied by increased D-frames in the area identified as normal on computed tomography images. Furthermore, measurement of DLD was very effective for evaluating diaphragmatic kinetics. CONCLUSIONS: This is a rapid and simple method for evaluation of respiratory kinetics for pulmonary diseases, which can reveal abnormalities in diaphragmatic kinetics and regional lung ventilation. Furthermore, quantification and visualization of respiratory kinetics is useful as an aid in interpreting dynamic chest radiographs.

Adult↗