Biomedical subjects

Takashi Imai

Publications and source records attributed to Takashi Imai.

At least 37 records · Page 2Linked to original sources

PubMed2005

A fast, simple method for screening radiation susceptibility genes by RNA interference.

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes.

Cell Count↗

PubMed2005

Molecular markers predicting radiotherapy response: report and recommendations from an International Atomic Energy Agency technical meeting.

PURPOSE: There is increasing interest in radiogenomics and the characterization of molecular profiles that predict normal tissue and tumor radioresponse. A meeting in Amsterdam was organized by the International Atomic Energy Agency to discuss this topic on an international basis. METHODS AND MATERIALS: This report is not completely exhaustive, but highlights some of the ongoing studies and new initiatives being carried out worldwide in the banking of tumor and normal tissue samples underpinning the development of molecular marker profiles for predicting patient response to radiotherapy. It is generally considered that these profiles will more accurately define individual or group radiosensitivities compared with the nondefinitive findings from the previous era of cellular-based techniques. However, so far there are only a few robust reports of molecular markers predicting normal tissue or tumor response. RESULTS: Many centers in different countries have initiated tissue and tumor banks to store samples from clinical trials for future molecular profiling analysis, to identify profiles that predict for radiotherapy response. The European Society for Therapeutic Radiology and Oncology GENEtic pathways for the Prediction of the effects of Irradiation (GENEPI) project, to store, document, and analyze sample characteristics vs. response, is the most comprehensive in this regard. CONCLUSIONS: The next 5-10 years are likely to see the results of these and other correlative studies, and promising associations of profiles with response should be validated in larger definitive trials.

Biomarkers, Tumor↗

PubMed2005

Partial molar volume of proteins studied by the three-dimensional reference interaction site model theory.

The three-dimensional reference interaction site model (3D-RISM) theory is applied to the analysis of hydration effects on the partial molar volume of proteins. For the native structure of some proteins, the partial molar volume is decomposed into geometric and hydration contributions using the 3D-RISM theory combined with the geometric volume calculation. The hydration contributions are correlated with the surface properties of the protein. The thermal volume, which is the volume of voids around the protein induced by the thermal fluctuation of water molecules, is directly proportional to the accessible surface area of the protein. The interaction volume, which is the contribution of electrostatic interactions between the protein and water molecules, is apparently governed by the charged atomic groups on the protein surface. The polar atomic groups do not make any contribution to the interaction volume. The volume differences between low- and high-pressure structures of lysozyme are also analyzed by the present method.

Binding Sites↗

PubMed2005

Hydrophobic effects on partial molar volume.

The hydrophobic effects on partial molar volume (PMV) are investigated as a PMV change in the transfer of a benzenelike nonpolar solute from the nonpolar solvent to water, using an integral equation theory of liquids. The volume change is divided into two effects. One is the "packing" effect in the transfer from the nonpolar solvent to hypothetical "nonpolar water" without hydrogen bonding networks. The other is the "iceberg" effect in the transfer from nonpolar water to water. The results indicate that the packing effect is negative and a half compensated by the positive iceberg effect. The packing effect is explained by the difference in the solvent compressibility. Further investigation shows that the sign and magnitude of the volume change depend on the solute size and the solvent compressibility. The finding gives a significant implication that the exposure of a hydrophobic residue caused by protein denaturation can either increase or decrease the PMV of protein depending on the size of the residue and the fluctuation of its surroundings.

Journal Article↗

PubMed2005

Resistance of regulatory T cells to glucocorticoid-induced [corrected] TNFR family-related protein (GITR) during Plasmodium yoelii infection.

CD4+ T cells are the major effector T cells against blood-stage Plasmodium yoelii infection. On the other hand, the lethal strain of P. yoelii (PyL) has acquired an escape mechanism from host T cell immunity by activating CD4+CD25+ regulatory T cells (Treg). Although the activation of Treg during PyL infection precludes the clearance of PyL from mice, it remains unclear whether activation of Treg is attributable to a specific response against PyL infection. Thus, we examined here whether Treg proliferate in an antigen-dependent manner during PyL infection. We also investigated the effector and regulatory mechanisms of Treg. Infection with PyL increased the number of CD4+CD25+ T cells, in which expression of Foxp3 mRNA is up-regulated. The Treg that were transferred into mice infected with PyL, but not with a non-lethal strain of P. yoelii (PyNL), proliferated during the initial 5 days following infection. The Treg from PyL-infected mice showed strong suppression compared with those from naive or PyNL-infected mice, and could suppress T cell activation by recognizing PyL- but not PyNL-derived antigens. Furthermore, the suppressive function of Treg activated in PyL-infected but not in naive mice could not be inhibited by treatment with an anti-glucocorticoid-induced TNFR family-related protein (GITR) mAb. These findings indicate that PyL infection specifically activates Treg that are specific for PyL-derived antigens. The infection also induces resistance for Treg to GITR signaling, and this eventually contributes to the escape of parasites from host T cell immunity.

Animals↗

PubMed2005

Inter-strain variance in late phase of erythematous reaction or leg contracture after local irradiation among three strains of mice.

AIM: To gain insights into inter-strain differences in radiosensitivity. METHODS: Mice of inbred strains, A/J, C57BL/6J, and C3H/HeMs, were irradiated at graded doses ranging from 20 to 60 Gy. Skin reaction and leg contraction were observed for a period of 230 days and between 175 and 350 days, respectively. Gene expressions in leg skin tissue were quantified by quantitative RT-PCR assay at 1, 12 and 72 h after 30 Gy irradiation. Mice were locally irradiated by using a Cs-137 source. RESULTS: The three strains showed various degrees of susceptibility to irradiation has evaluated by skin scores. Large inter-strain differences were also detected in the lengths of contraction. Expressions of several genes such as Per3 and Rad51ap1 displayed inter-strain differences. CONCLUSIONS: The continuum model of tissue injury revealed that genetic factor, which varies among strains, is one of the causes of variances in severity of damage after irradiation.

Animals↗

PubMed2005

Fine mapping of radiation susceptibility and gene expression analysis of LEC congenic rat lines.

LEC rats constitute an animal model of high susceptibility to X-rays. We developed congenic LEC rat lines (recipient strain, Fischer 344 (F344)) and performed genome-wide genotyping to identify radiation susceptibility genes. We mapped seven positional candidate genes, Bmp10, Gpr73, Gp9, Cnbp, Copg, Rab7, and Rpn1, to an approximately 1.2-Mb region located between loci D4Got85 and D4Got148 on chromosome 4. None of the seven genes has been reported to be associated with radiation susceptibility. Comparison of the coding sequences for these seven genes in F344 and LEC rats showed no changes in deduced amino acid sequences. We determined gene expression differences in Gp73, Gp9, and Cnbp as well as strain-specific variations in upstream sequences of these genes. Our results suggest that radiation susceptibility in the LEC rat is primarily attributable to one of the genes within this approximately 1.2-Mb region; however, expression analysis gave no clear indication as to which gene is responsible.

Animals↗

PubMed2005

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines.

The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2005

Potential in a single cancer cell to produce heterogeneous morphology, radiosensitivity and gene expression.

Morphologically heterogeneous colonies were formed from a cultured cell line (KYSE70) established from one human esophageal carcinoma tissue. Two subclones were separated from a single clone (clone13) of KYSE70 cells. One subclone (clone13-3G) formed mainly mounding colonies and the other (clone 13-6G) formed flat, diffusive colonies. X-irradiation stimulated the cells to dedifferentiate from the mounding state to the flat, diffusive state. Clone 13-6G cells were more radiosensitive than the other 3 cell lines. Clustering analysis for gene expression level by oligonucleotide microarray demonstrated that in the radiosensitive clone13-6G cells, expression of genes involved in cell adhesion was upregulated, but genes involved in the response to DNA damage stimulus were downregulated. The data demonstrated that a single cancer cell had the potential to produce progeny heterogeneous in terms of morphology, radiation sensitivity and gene expression, and irradiation enhanced the dedifferentiation of cancer cells.

Cell Differentiation↗

PubMed2005

Strain-dependent differences in locomotor activity after local brain irradiation with 30 GyE of carbon ions.

This study investigated strain differences in brain damage among male A/J, C57BL/6JNrs and C3H/HeNrs mice after local brain irradiation. Whole brains were irradiated with a single dose of 30 GyE carbon ion beams and then locomotor activity was determined as body heat of each animal. The daily locomotor activities of untreated mice differed among strains. Non-irradiated C57BL/6JNrs mice were more active than A/J mice. This variance became more obvious immediately after irradiation, when the activity of A/J and C3H/HeNrs mice diminished, whereas that of C57BL/6JNrs mice increased at the beginning of the active phase and remained elevated for three days after irradiation. The altered activities of all three strains of irradiated mice gradually recovered to normal within three to four days.

Animals↗

PubMed2004

Statistical sequence analyses of G-protein-coupled receptors: structural and functional characteristics viewed with periodicities of entropy, hydrophobicity, and volume.

G-protein-coupled receptors (GPCRs) play a crucial role in signal transduction and receive a wide variety of ligands. GPCRs are a major target in drug design, as nearly 50% of all contemporary medicines act on GPCRs. GPCRs are membrane proteins possessing a common structural feature, seven transmembrane helices. In order to design an effective drug to act on a GPCR, knowledge of the three-dimensional (3D) structure of the target GPCR is indispensable. However, as GPCRs are membrane bound, their 3D structures are difficult to obtain. Thus we conducted statistical sequence analyses to find information about 3D structure and ligand binding using the receptors' primary sequences. We present statistical sequence analyses of 270 human GPCRs with regard to entropy (Shannon entropy in sequence alignment), hydrophobicity and volume, which are associated with the alpha-helical periodicity of the accessibility to the surrounding lipid. We found periodicity such that the phase changes once in the middle of each transmembrane region, both in the entropy plot and in the hydrophobicity plot. The phase shift in the entropy plot reflects the variety of ligands and the generality of the mechanism of signal transduction. The two periodic regions in the hydrophobicity plot indicate the regions facing the hydrophobic lipid chain and the polar phospholipid headgroup. We also found a simple periodicity in the plot of volume deviation, which suggests conservation of the stable structural packing among the transmembrane helices.

Entropy↗

PubMed2004

Temperature and pressure effects on conformational equilibria of alanine dipeptide in aqueous solution.

We investigated the temperature and pressure effects on conformational equilibria of N-acetyl-L-alanine-N'-methylamide (AAlaMA) in aqueous solution by Raman spectroscopy. Scattering intensities in the skeletal stretching mode of AAlaMA in aqueous solution were decomposed into some component bands by the spectra analysis. Our results indicate that each component band for AAlaMA adopts not only the P(II) and alpha(R) conformations but also the C(7eq) conformation. From temperature and pressure dependencies of the band intensities, we determined the enthalpy differences and the volume differences between the conformers. The C(7eq) conformer is enthalpically most stable due to the intramolecular hydrogen bond. The partial molar volume of the C(7eq) conformer is the smallest through the solvent-exclusion effect rather than the solute-solvent electrostatic interaction effect.

Alanine↗

PubMed2004

Detection of pairwise residue proximity by covariation analysis for 3D-structure prediction of G-protein-coupled receptors.

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.

Animals↗

PubMed2004

Effects of carbon-ion beams on human pancreatic cancer cell lines that differ in genetic status.

The relative biologic effectiveness (RBE) of carbon-ion beams at 3 different linear energy transfer (LET) values (13, 50, and 80 keV/microm) accelerated by the Heavy Ion Medical Accelerator in Chiba on human pancreatic cancer cell lines differing in genetic status was determined. The RBE values were calculated as D10, the dose (Gy) required to reduce the surviving fraction to 10%, relative to X-rays. We also investigated apoptosis and the relationship between D10 and the cell cycle checkpoint using morphologic examination and flow cytometry analysis, respectively. The RBE values calculated by the D10 values ranged from 1.16 to 1.77 for the 13-keV/microm beam and from 1.83 to 2.46 for the 80-keV/microm beam. A correlation between the D10 values of each cell line and intensity of G2/M arrest was observed. In contrast, LET values did not clearly correlate with induction of apoptosis. These results suggest that carbon-ion beam therapy is a promising modality. Elucidation of the mechanisms of G2/M arrest and apoptosis may provide clues to enhancing the effects of radiation on pancreatic cancer.

Apoptosis↗

PubMed2004

Strain dependent differences in a histological study of CD44 and collagen fibers with an expression analysis of inflammatory response-related genes in irradiated murine lung.

Using a mouse model, we investigated the mechanisms of heterogeneity in response to ionizing radiation in this research. C57BL/6J and C3H/HeMs mice were irradiated with gamma rays at 10 and 20 Gy. The animals were sacrificed at times corresponding to the latent period, the pneumonic phase, and the start of the fibrotic phase for histological investigation. Small areas of fibrosis initially appeared in C57BL/6J mice at 4 weeks postirradiation with 20 Gy, whereas small inflammatory lesions appeared at 4 and 8 weeks after 20 and 10 Gy, respectively. The alveoli septa were thickened by an infiltration of inflammatory cells, and alveoli were obliterated in lungs from C57BL/6J mice after 20 Gy irradiation. At 24 hours and from 2 to 4 weeks postirradiation, fourfold more CD44 positive cells had accumulated in the lungs of C3H/HeMs than in C57BL/6J mice. Hyaluronan accumulated 12 hours after irradiation, and the rapid resolution was achieved within 2 weeks in the lungs in both strains of mice. C57BL/6J mice lungs accumulated dense collagen at 8 weeks. Quantitative RT-PCR assay was performed for several genes selected by cDNA microarray analysis. The expression of several genes, such as Cap1, Il18, Mmp12, Per3, Ltf, Ifi202a, and Rad51ap1 showed strain-dependent variances. In conclusion, a histological investigation suggested that C3H/HeMs mice were able to induce a more rapid clearance of matrix after irradiation than C57BL/6J mice. The expression analysis showed that the several genes are potentially involved in interstrain differences in inflammatory response causing radiation-induced lung fibrosis.

Animals↗

PubMed2004

Fractionated irradiation augments inter-strain variation of skin reactions among three strains of mice.

The multifraction regimens commonly used in conventional clinical radiotherapy are largely based on radiobiological experiments. However, no experimental reports on skin reactions focusing on inter-strain differences have displayed clinical relevance to the fractionated dose schedule. In this study, mice of inbred strains A/J, C57BL/6J, and C3H/HeMs were used to reveal inter-strain difference after multifractionated irradiation. Irradiation was performed daily at graded doses of 30-60 Gy total doses, with 10 fractions of 3-6 Gy. Acute skin reactions following irradiation were scored for 50 days after irradiation. Dividing a dose into a number of fractions obviously spared skin damage in the three strains of mice. No mouse exhibited a skin damage score more than 1.5, while single dose irradiation resulted in skin damage scores up to 3. The three different strains, however, showed varying susceptibility to fractionated irradiation within the range under 1.5. C3H/HeMs did not display any skin reaction after irradiation with 40 Gy total dose, while C57BL/6J and A/J demonstrated various skin reactions. Different latent periods of damage were also observed among the strains after irradiation at each dose. Our data suggest that genetic factors cause obvious variations in severity of damage and latent period after fractionated irradiation.

Animals↗

PubMed2004

Radiosensitivity of peripheral blood lymphocytes obtained from patients with cancers of the breast, head and neck or cervix as determined with a micronucleus assay.

The in vitro radiation sensitivities of peripheral blood lymphocytes obtained from 48 normal females and 168 female cancer patients were measured with the cytokinesis-blocking micronucleus assay. Cancer patients group had significantly higher mean baseline micronucleus frequency than normal healthy controls. Breast cancer patients were more radiosensitive than normal individuals. Cervical cancer cases were less radiation sensitive than normal subjects. The relative lack of radiation sensitivity in cervical-cancer cases could be due to modification of the radiosensitivity of patients' immune-responsible cells by human papillomaviruses infection. Normal individuals and cancer patients were classified according to their radiation sensitivity which was evaluated with the radiation-induced micronucleus frequencies. Such a classification will be an important initial step to characterize the radiosensitive, radioresistant, or cancer-prone individuals using specific SNP typing.

Adult↗

PubMed2003

Beneficial effects of apolipoprotein A-I on endotoxemia.

PURPOSE: Although many studies have shown the beneficial effects of lipoproteins on animals with endotoxemia, little is known about the impact of apolipoprotein A-I (apoA-I) on tumor necrosis factor Alpha (TNF-Alpha) release in response to lipopolysaccharide (LPS). The present study was conducted to determine whether the administration of apoA-I inhibits the release of TNF-Alpha and influences the survival rate of rats with endotoxemia. METHODS: Forty male Wistar rats were divided randomly into four groups. Rats in the first and second groups were given 1 mg/kg LPS intraperitoneally (i.p.) and blood was collected 1 h later to measure the serum levels of TNF-Alpha. Either 10 mg/kg apoA-I or Tris-buffered saline was injected i.p. and the serum TNF-Alpha levels were measured again 2 h later. Rats in the third and fourth groups were given 5 mg/kg LPS. Following the administration of 10 mg/kg apoA-I or Tris-buffered saline, animals were observed for 5 days and survival rates were determined. RESULTS: ApoA-I inhibited the release of serum TNF-Alpha and improved the survival rates of rats with endotoxemia. CONCLUSION: The administration of apoA-I suppressed the TNF-Alpha release in endotoxemia and decreased the mortality rates of rats.

Animals↗