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Hiroshi Kobayashi

Publications and source records attributed to Hiroshi Kobayashi.

At least 19 recordsLinked to original sources
PubMed2026

PDGFRA amplification could be a poor prognostic factor of advanced undifferentiated pleomorphic sarcoma in a comprehensive genomic profiling cohort.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) is the most common pleomorphic sarcoma, and its genomic landscape has been analysed, albeit in small numbers. This study aimed to clarify the relationship between gene variants and the prognosis of patients with advanced UPS. METHODS: This retrospective cohort study was conducted to analyse the data of patients with advanced UPS using a registry of the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database up to Oct 2025 in Japan, analysed using comprehensive genomic profiling assay. RESULTS: A total of 233 patients with advanced UPS were identified in the C-CAT database; 151 men (64.8%), median age: 60.2&#xa0;years. TP53 variant (55%) was the most frequent event and the rate of Platelet-derived growth factor receptor alpha (PDGFRA) and KDR amplification were 9% and 6%, respectively. PDGFRA amplification co-occurred with KDR amplification (P&#xa0;<&#xa0;0.001). Survival from the initiation of chemotherapy was analysed by adjusting for length bias inherent in the database using the Kaplan-Meier estimator, an established method of adjustment. Patients with PDGFRA amplification (11 patients) had a worse prognosis than those without PDGFRA amplification [hazard ratio 2.9, 95% confidence interval 1.2-6.9 (P&#xa0;=&#xa0;0.02)]. TP53 alterations (P&#xa0;=&#xa0;0.24) were not associated with prognosis. In addition, treatment time with pazopanib with PDGFRA amplification [4 patients, 2.3&#xa0;months (1.2-11.2&#xa0;months)] was not different with those without PDGFRA amplification [28 patients, 3.8&#xa0;months (0.9&#xa0;months-not reached)] (P&#xa0;=&#xa0;0.52). CONCLUSIONS: For patients with advanced UPS, PDGFRA amplification was a poor prognostic factor and is not related to the efficacy of pazopanib treatment.

PDGFRA amplification↗
PubMed2026

Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.

Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.

lentiviral vector↗
PubMed2007

RNA expression in a cartilaginous fish cell line reveals ancient 3' noncoding regions highly conserved in vertebrates.

We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3' UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3' mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.

3' Untranslated Regions↗
PubMed2006

Particle simulation approach for subcellular dynamics and interactions of biological molecules.

BACKGROUND: Spatio-temporal dynamics within cells can now be visualized at appropriate resolution, due to the advances in molecular imaging technologies. Even single-particle tracking (SPT) and single fluorophore video imaging (SFVI) are now being applied to observation of molecular-level dynamics. However, little is known concerning how molecular-level dynamics affect properties at the cellular level. RESULTS: We propose an algorithm designed for three-dimensional simulation of the reaction-diffusion dynamics of molecules, based on a particle model. Chemical reactions proceed through the interactions of particles in space, with activation energies determining the rates of these chemical reactions at each interaction. This energy-based model can include the cellular membrane, membranes of other organelles, and cytoskeleton. The simulation algorithm was tested for a reversible enzyme reaction model and its validity was confirmed. Snapshot images taken from simulated molecular interactions on the cell-surface revealed clustering domains (size approximately 0.2 microm) associated with rafts. Sample trajectories of raft constructs exhibited "hop diffusion". These domains corralled the diffusive motion of membrane proteins. CONCLUSION: These findings demonstrate that our approach is promising for modelling the localization properties of biological phenomena.

Biopolymers↗
PubMed2006

Performance of monolithic silica capillary columns with increased phase ratios and small-sized domains.

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of approximately 1.3 microm and silica skeletons of approximately 0.9 microm) showed a plate height of less than 5 microm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of approximately 1, and approximately 7 microm at u = 8 mm/s. With a permeability similar to that of a column packed with 5-microm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-microm particles, and showed performance in the "forbidden region" for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15,000-30,000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.

Benzene Derivatives↗
PubMed2006

Incorporation of nonionic emulsifiers inside particles in emulsion polymerization: mechanism and methods of suppression.

Emulsion polymerizations of styrene were carried out using two kinds of polyoxyethylene lauryl ether nonionic emulsifiers having different hydrophilic-lipophilic balances (HLB): Emulgen 109P (HLB 13.6); and Emulgen 150 (HLB 18.3). In both cases, incorporation of emulsifier inside polystyrene (PS) particles was clearly observed, as previously reported for the emulsion polymerization of styrene and methacrylic acid using polyoxyethylene nonyl phenyl ether (Emulgen 911, HLB 13.7) nonionic emulsifier. The generality of the incorporation phenomenon of nonionic emulsifier inside polymer particles in emulsion polymerization was clarified. In the case of Emulgen 109P, which is more hydrophobic than Emulgen 150, about 30% of the total amount was incorporated inside the PS particles, higher than for Emulgen 150 (15%). The difference seemed to be ascribed to the difference in the affinities between the nonionic emulsifiers and styrene, which cause the incorporation of emulsifier. On the basis of this idea, suppression of the incorporation was achieved by decreasing the polymerization temperature and the monomer-feed rate. This strongly supports the proposed incorporation mechanism.

Journal Article↗
PubMed2006

Batchwise assessment of porcine embryos for cryotolerance.

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.

Animals↗
PubMed2006

[Estimation of operator dose by dose area product meter].

Because of the more advanced and more complex procedures in interventional radiology (IVR), longer treatment times have become necessary. Therefore, it is important to determine the exposure doses received by operators and patients. Operator doses arising from the use of X-rays are mainly due to scattered radiation. The purpose of this study was to assess the feasibility of estimating operator dose by dose area product (DAP), which shows the total X-ray output from the collimator. DAP showed a strong correlation with the space dose from the fundamental examination. In clinical practice, we measured the exposure doses of the neck, left shoulder, left hand, and right finger using a thermoluminescence dosimeter (TLD). These then were compared with the DAP. The results indicated that the dose equivalents (H70 microm) of the neck and left shoulder were strongly correlated with DAP (r=0.85, 0.86), whereas the H70 microm of the left hand and right finger were less closely correlated (r=0.40, 0.48). In comparison with the fluoroscopic time, the dose equivalents showed a better correlation with DAP in all the evaluated parts. The effective doses for the operator were strongly correlated with DAP (r=0.87). When measurements are not available, dose equivalents and operator effective doses can be estimated by the DAP, as indicated by the strong correlations recognized in this study.

Computer Systems↗
PubMed2006

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias.

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.

Animals↗
PubMed2006

The role of intravenous coronary thrombolysis for patients with acute myocardial infarction in different treatment strategies.

OBJECTIVE: To examine acute-phase outcomes in acute myocardial infarction (AMI) according to different initial treatments. PATIENTS AND METHODS: This retrospective study involved 405 patients with AMI who had undergone coronary angiography during the acute phase. The patients were retrospectively examined by dividing into groups according to treatment received: intravenous coronary thrombolysis (IVCT) (n=83), intracoronary thrombolysis (ICT) (n=62), and percutaneous coronary intervention (PCI) (n=221). RESULTS: TIMI 3 flow at the initial angiography was higher in the IVCT group (P<0.05) at 32.5% in the IVCT group and 21.7% in the non-IVCT group. The time from onset to initiation of treatment was shorter in the IVCT group (P<0.001) at 227 min in the IVCT group, 337 min in the ICT group, and 479 min in the PCI group. The acute-phase mortality was lower in the IVCT group (P<0.05) at 2.4% in the IVCT group, 3.2% in the ICT group, and 11.8% in the PCI group. According to sub-analysis, the restenosis rate during the chronic phase after PCI did not differ with or without antecedent administration of a thrombolytic agent. CONCLUSION: IVCT as an initial treatment for AMI enabled the fastest reperfusion at TIMI > or = 2 flow, resulting in a good acute-phase outcome.

Aged↗
PubMed2006

Potassium/proton antiport system of Escherichia coli.

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.

Base Sequence↗
PubMed2006

Enhanced spontaneous metastasis in bikunin-deficient mice.

Previously, we showed that bikunin, a Kunitz-type protease inhibitor, inhibits invasion and metastasis in several types of cancer cells possibly through suppression of upregulation of urokinase-type plasminogen activator (uPA) expression. Bikunin corresponds to a light chain of the inter-alpha inhibitor. To explore critical role of endogenous bikunin, we used bikunin knockout (Bik-/-) mice. Here, we show that 1) higher frequency of spontaneous 3LL lung metastasis was observed in Bik-/- mice compared to Bik+/+ mice, suggesting that bikunin deficiency increases the sensitivity of mice to lung metastasis; 2) administration of exogenous bikunin caused a significant reduction of lung metastasis in Bik-/- and Bik+/+ mice; 3) primary and metastatic tumors significantly upregulated uPA and PAI-1 expression in Bik-/- mice relative to Bik+/+ mice at least through phosphorylation of ERK1/2 and 4) exogenous bikunin suppressed phosphorylation of ERK1/2 and upregulation of uPA and PAI-1 expression in 3LL cells in response to G-CSF. These data allow us to conclude that the increased sensitivity of Bik-/- mice to lung metastasis in vivo is due to a lack of circulating proteins of the inter-alpha inhibitor family, especially bikunin.

Animals↗
PubMed2006

Annexin 2 and hemorrhagic disorder in vascular intimal carcinomatosis.

Vascular intimal carcinomatosis refers to a characteristic tumor proliferation on vascular intima that replaces normal endothelium. This pathological event of unknown cause is quite different from tumor thrombotic microangiopathy due to the absence of thrombi on the tumor cell surfaces. We analyzed renal transitional cell carcinoma cases with metastasis to the main pulmonary arteries and marked hyperfibrino(geno)lysis. The fibrinogen-derived products from patients' plasma were identified as D1A/gamma, D1/gamma, and D1/beta by immunoblotting with the NH2-terminus of the fragment D specific antibody JIF-23. In all cases, the neoplastic cells with vascular intimal carcinomatosis were stained positive for anti-human annexin 2, which is a unique cell surface co-receptor for plasminogen and tissue-type plasminogen activator. In contrast, normal renal pelvic mucosa or renal transitional cell carcinoma without vascular intimal carcinomatosis did not express any annexin 2. The isolated transitional cell carcinoma cells contained annexin 2 mRNA and expressed its protein. Anti-annexin 2 antibody and transfection of annexin 2 small interfering RNA into these carcinoma cells significantly inhibited tissue-type plasminogen activator dependent plasmin generation. These findings suggest that annexin 2 mediated fibrinolysis on the transitional cell carcinoma cells may play a role in inducing hemorrhagic disorder in vascular intimal carcinomatosis.

Adult↗
PubMed2006

Relationship between time elapsed after human chorionic gonadotropin administration and developmental stage in porcine embryos collected from prepubertal gilts.

We examined the relationship between the time elapsed after human chorionic gonadotropin (hCG) administration and developmental stage of porcine embryos after collection. Prepubertal gilts, 7 to 8 months old, were given 1500 IU equine chorionic gonadotropin (eCG) intramuscularly, followed by 500 IU hCG 72 h later. The treated gilts were inseminated artificially on Day 1 (Day 0=the day of hCG administration) and on Day 2. Embryos were collected surgically on Day 6 (140, 144, and 147 h after hCG administration) or on Day 7 (164, 168, and 171 h), and the developmental stages of the collected embryos were examined. From 75.2% (276/367) of the prepubertal gilts treated with hormones, we collected an average of 20.7 embryos per gilt with normal morphology. At 140 h after hCG administration, morulae (54.4%) could be collected. At 144 h, morulae and early blastocysts (57.7% and 28.9%, respectively) were collected. By 147 h, the proportion of embryos at the blastocyst to expanded blastocyst stages had increased (10.0%). From 164 h to 171 h, expanding or expanded blastocysts of more than 200 microm in diameter and hatched blastocysts could be collected. The proportion of hatched blastocysts increased from 3.2% (164 h) to 41.0% (171 h). These results suggests that although the number of ovulations differed among gilts, porcine embryos at the appropriate stages can be collected efficiently by controlling the time elapsed between hCG administration and embryo collection.

Animals↗
PubMed2006

Requirement of an IkappaB-beta COOH terminal region protein for acidic-adaptation in CHO cells.

We previously reported that an IkappaB-beta COOH terminal region protein (designated CTIB) was essential for the proliferation of CHO cells under acidic stress (Lao et al., 2005. J Cell Physiol 203(1):186-192). In order to investigate the mechanisms underlying the requirement of CTIB for acidic adaptation, CTIB was silenced with an RNAi technique in CHO cells. CTIB silencing resulted in those cells completely failing to proliferate and maintain intracellular pH (pHi) homeostasis at an extracellular pH (pHe) of 6.3. An increased activation of p38 MAP kinase was induced by CTIB silencing at the low pH value. CTIB was only present in the cytoplasm and co-immunoprecipitation of the cytoplasmic fraction revealed that the loss of CTIB led to a loss of p65 in the immunoprecipitate complex. CTIB silencing reduced both the decrease in p65 and the increase in p50 in the nucleus when the cells were incubated at pHe 6.3. In cells with CTIB silenced, the transcriptions of p65, p105, and IL1-beta were suppressed, and decreases in both the transcription and activity of MnSOD were observed at pHe 6.3. Suppression of these genes suggested a suppressed NF-kappaB activity since p105, IL1-beta, and MnSOD were target genes of NF-kappaB. Our data demonstrated that CTIB functioned to prevent the over-accumulation of p65 in the nucleus, ensuring the appropriate composition of the NF-kappaB complex in the nucleus to respond to stimuli under acidic conditions.

Acids↗
PubMed2006

A kinetic parameter concerning mass transfer in silica monolithic and particulate stationary phases measured by the peak-parking and slow-elution methods.

Mass transfer in monolithic C18-silica stationary phases and C18-silica gel particles was studied. A traditional kinetic parameter, gamma(s)D(s), which is a diffusion coefficient of solute molecules in the stationary phase, was measured by two unusual approaches, i.e., peak-parking and slow-elution methods. The correlation between the ratio of gamma(s)D(s) to molecular diffusivity (Dm) and the retention factor (k) was represented by one common curve, irrespective of the RPLC conditions. A similar curved profile was also observed between another kinetic parameter (D(Ls)), which is related to the axial diffusive molecular migration in the stationary phase, and the retention equilibrium constant (Ka). The values of D(Ls) and Ka were calculated from those of gamma(s)D(s) and k, respectively. The ratio of D(Ls)/Dm increases with decreasing Ka and seems to approach around unity when Ka is infinitely small. The dependence of D(Ls) on Ka was also studied from extra-thermodynamic points of view. The linear correlation between In D(Ls) and In Ka suggests the existence of a kind of linear free energy relationship between the mass transfer in the stationary phase and the retention equilibrium. Because these characteristics of D(Ls) are similar to those of the surface diffusion coefficient (D(sur)), D(Ls) seems to correspond to D(sur).

Chemistry Techniques, Analytical↗
PubMed2006

Systemic sclerosis and pseudomesotheliomatous adenocarcinoma of the lung.

A 55-year-old man, diagnosed with systemic sclerosis (SSc) for 20 years, was admitted to our hospital for exertional dyspnea and pleural effusion. Computed tomography scan and cytological findings of the pleural fluid suggested malignant mesothelioma. In the postmortem examination, the tumor was pathologically diagnosed as pseudomesotheliomatous adenocarcinoma (PMA) of the lung, classified into pleomorphic carcinoma with adenocarcinoma component according to the new World Health Organization guidelines. This is the first case report of SSc with PMA.

Adenocarcinoma↗