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Biomedical subjects

H Tauchi

Publications and source records attributed to H Tauchi.

At least 19 recordsLinked to original sources

Accumulation of cells at G2/M stage by low dose-rate irradiation renders the cell population more susceptible to the subsequent induction of 6-thioguanine-resistant mutations by 252Cf fission neutrons.

A previous study on mutagenesis by 252Cf radiation in mouse L5178Y cells showed that the frequency was higher when the dose was delivered chronically, which was in sharp contrast to the results by gamma-rays (Nakamura and Sawada, 1988). A subsequent study using synchronized cells revealed that the cells at G2/M stage were uniquely sensitive to mutation induction by 252Cf radiation but not so by gamma-rays (Tauchi et al, 1993). We carried out the present study to test the possibility that radiation-induced G2 block may be a major determinant of the inverse dose-rate effect following chronic 252Cf radiation. Growing cell population was first subjected to conditioning gamma or 252Cf radiation with different dose-rates, followed by cell cycle distribution analysis and 252Cf mutagenesis. We found that G2/M fraction increased by 3- to 4-fold when the conditioning doses (2 Gy of gamma or 1 Gy of 252Cf radiation) were delivered chronically over 10 hours but only slightly so when the same doses were delivered for 1 hour or less. Subsequent 252Cf irradiation gave higher mutation frequencies in the cells pre-irradiated with gamma-rays over a protracted period of time than in those with higher dose-rate gamma-rays. These results suggest that radiation-induced G2 block would be at least partly (but can not be totally) responsible for the inverse dose-rate effect.

Animals

Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation.

Vascular endothelial growth factor (VEGF) has been identified as a peptide growth factor specific for vascular endothelial cells. In this study, we demonstrated the expression of the KDR gene transcript, which encodes a cell surface receptor for VEGF, in normal human hematopoietic stem cells, megakaryocytes, and platelets as well as in human leukemia cell lines, HEL and CMK86. Moreover, we showed the expression of VEGF gene transcript in these normal fresh cells and cell lines. To elucidate biological functions of VEGF on hematopoiesis, we determined whether this growth factor has mitogenic activity to hematopoietic cells or the ability to suppress apoptotic cell death. The liquid culture and colony-formation assay revealed that VEGF suppressed apoptotic cell death of both CMK86 cells and normal hematopoietic stem cells caused by gamma-ray irradiation, although mitogenic activity of VEGF was not detected. The ability of VEGF to suppress apoptotic cell death was independent of the change of cell cycle distribution. These data suggest that VEGF may play an important role in survival or maintenance of hematopoietic stem cells due to the prevention of apoptotic cell death caused by some stresses such as ionizing radiation and that VEGF may give leukemia cells some abilities of resistance against radiotherapy in an autocrine or paracrine manner.

Apoptosis

Radiation-induced apoptotic cell death in human gastric epithelial tumour cells; correlation between mitotic death and apoptosis.

The mode of cell death in cells undergoing mitotic death after gamma-irradiation was studied in seven human gastric epithelial tumour cell lines and two strains of normal gastric fibroblasts. Apoptotic cells were frequently observed in all tumour lines after irradiation, whereas the two fibroblast strains were quite low in apoptosis frequency. The advent of apoptosis depended on the radiation doses and incubation time. Detailed analysis of one of the carcinoma lines, SH101-P4, revealed that G2-phase arrest was maximum at 12 h postirradiation. The cells began to escape G2 arrest by 24 h. Apoptotic cells began to increase at 12 h postirradiation and became maximal from 72 to 96 h. Apoptosis developed in the G1 phase of the cell cycle subsequent to the irradiation. These results suggest that apoptosis is one of the modes of mitotic death after irradiation.

Adenocarcinoma

Disappearance of neutrophil fluctuations in a child with cyclic neutropenia by combination therapy of granulocyte colony-stimulating factor and high-dose immunoglobulin.

We report on a patient with cyclic neutropenia who was treated by granulocyte colony-stimulating factor (G-CSF) and high-dose immunoglobulin. The serial examination revealed cyclic fluctuations in the numbers of neutrophils, monocytes, platelets in peripheral blood, and in the serum G-CSF concentration. Bone marrow examination confirmed a cyclic fluctuation of both progenitor cells (CFU-GM) and CD10-positive B cells. The therapy of G-CSF followed by high-dose immunoglobulin achieved a disappearance of neutrophil oscillations. It suggested that the combination therapy of G-CSF with high-dose immunoglobulin might be effective for cyclic neutropenia.

B-Lymphocyte Subsets

Neutron generator at Hiroshima University for use in radiobiology study.

A neutron generator (HIRRAC) for use in radiobiology study has been constructed at the Research Institute for Radiation Biology and Medicine, Hiroshima University (RIRBM). Monoenergetic neutrons of which energy is less than 1.3 MeV are generated by the 7Li(p,n)7 Be reaction at proton energies up to 3 MeV. The protons are accelerated by a Schenkel-type-accelerator and are bombared onto the 7Li-target. An apparatus for the irradiation of biological material such as mice, cultured cells and so on, was designed and will be manufactured. Neutron and gamma-ray dose rates were measured by paired (TE-TE and C-CO2) ionization chambers. Contamination of the gamma ray was less than about 6% when using 10-microns-thick 7Li as a target. Maximum dose rates for the tissue equivalent materials was 40 cGy/min at a distance of 10 cm from the target. Energy distributions of the obtained neutrons have been measured by a 3He-gas proportional counter. The monoenergetic neutrons within an energy region from 0.1 to 1.3 MeV produced by thin 7Li or 7LiF targets had a small energy spread of about 50 keV (1 sigma width of gaussian). The energy spread of neutrons was about 10% or less at an incident proton energy of 2.3 MeV. We found that HIRRAC produces small energy spread neutrons and at sufficient dose rates for use in radiobiology studies.

Animals

Analysis of mitotic cell death caused by radiation in mouse leukaemia L5178Y cells: apoptosis is the ultimate form of cell death following mitotic failure.

The appearance of various abnormal cells after irradiation was investigated in growing mouse leukaemia L5178Y cells. Morphologically defined apoptotic cells started to emerge at 10 h after irradiation and the frequency reached a peak at around 48 h being similar to the frequency of other abnormal cells, i.e. micronucleated, multinucleated and giant cells. Necrotic cells were rarely seen. The frequency of apoptosis and other abnormal cells depended on the radiation dose. The typical DNA ladder pattern for apoptosis was observed in the agarose gel electrophoretic analysis of the cells at 24-96 h postirradiation. A decline in the frequency of apoptotic cells occurred with longer incubation, which was associated with a sharp increase in cloning efficiency. Changes in the growth rate of the irradiated cell population during the postirradiation period could be reasonably well described by a simple model using the frequencies of apoptosis and other abnormal cells. The results suggest that apoptosis is the ultimate form of cell death via mitotic failure caused by relatively small doses of radiation in L5178Y cells.

3T3 Cells

Suppression of gamma- and neutron-induced neoplastic transformation by ascorbic acid in Balb/c 3T3 cells.

The effects of ascorbic acid on the neoplastic transformation induced by 60Co gamma-rays or 252Cf fission neutrons were studied. The transformation induced by each of the radiations was markedly suppressed when ascorbic acid was added daily to the culture medium during the first 8 days of the post-irradiation period. If ascorbic acid was added for a total of 8 days as mentioned above, but with 1 day interruption in the middle, the suppression of transformation was decreased. Ascorbic acid also suppressed the promotion of radiation transformation by 12-O-tetradecanoylphorbol-13-acetate (TPA) when both chemicals were added to the medium together. However, promotion of radiation transformation by TPA was observed if both the chemicals were added sequentially during the post-irradiation period, i.e. ascorbic acid for the first 7 days followed by TPA. These results suggest that continuous administration of ascorbic acid for a certain number of days is needed to suppress the radiation transformation, and also suggest that it acts on the promotion stage of transformation.

3T3 Cells

Cell cycle dependence for the induction of 6-thioguanine-resistant mutations: G2/M stage is distinctively sensitive to 252Cf neutrons but not to 60Co gamma-rays.

Cell cycle dependence for the induction of 6-thioguanine-resistant mutation was investigated using synchronized mouse L5178Y cells after exposure to 4 Gy to 60Co gamma-rays or 1 Gy of 252Cf radiation (fission neutrons). Maximal mutation frequency was observed immediately after release from colcemid block (G2/M phase) after 252Cf radiation, whereas it was 1 h after the release (G1 phase) after 60Co gamma-rays. When the mutation frequency was plotted against the surviving fraction, a general correlation was observed between the two parameters except for G2/M and G1 phases which were more mutable per lethal event for 252Cf and 60Co radiations, respectively.

Animals

Age dependency of mitochondrial DNA decrease differs in different tissues of rat.

Mitochondrial DNA contents were measured using non-radioactive DNA probes for specific mtDNA sequences. The nuclear repetitive DNA sequence, LINE, was used as an internal standard marker. Age-dependent decreases in the mtDNA/nucDNA ratio in rat hepatic cells were confirmed with this method and, furthermore, a similar decrease was found in cardiac muscles. On the other hand, in thigh skeletal muscles such a decrease was not observed, slight increases being rather found in 18- to 24-month-old rats. The possible relationship of the decrease in mtDNA and the mechanism underlying animal senescence was discussed.

Aging

Quantitative immunohistochemistry of parvalbumin expression in the rat diaphragm.

Parvalbumin is a calcium-binding protein involved in the mechanism of Ca++ exchange between sarcoplasmic reticulum and the myofibrils. This characteristic promotes a direct correlation between the amount of parvalbumin and the velocity of contraction-relaxation of skeletal muscle fibers. To quantify the expression of parvalbumin in the rat diaphragm, transverse paraffin sections of this muscle were processed immunohistochemically using antiparvalbumin monoclonal antibody. The stained sections were inspected on an image analysis device to evaluate the intensity of the immunoreaction and to make morphologic measurements of the muscle fibers. The immunoreaction intensity data allowed the division of fibers in fast- and slow-twitch types. The rat diaphragm was characterized as a fast-twitch muscle due to the presence of 59.8% of fast-twitch fibers. The fibers' lesser diameter values showed that the slow-twitch fibers had a small diameter as compared to the fast-twitch ones (P < .001). The method used suggests that immunohistochemistry for parvalbumin assisted by image analysis is a feasible technique to separate fast- and slow-twitch skeletal muscle fibers in paraffin sections and to characterize their morphologic profiles.

Animals

Quantitation of changes in mitochondrial DNA during aging and regeneration of rat liver using non-radioactive DNA probes.

By using DNA probes prepared from cloned cells which contain mitochondrial DNA (mtDNA) sequences in plasmids, the specific detection of mtDNA became possible in the presence of large excess of nuclear DNA by DNA-DNA hybridization. For this purpose, we prepared mtDNA probes labeled with non-radioactive substrate, which allowed a wider possibility of application. This method revealed that the contents of mtDNA in rat liver are strikingly decreased during aging. Furthermore, it was observed that although mtDNA content increased upon partial hepatectomy even in old rats, it decreased to the pre-operation level rather rapidly within 1 week after reaching peak in regenerated liver.

Aging

Effect of TPA, okadaic acid and 1 alpha,25-dihydroxyvitamin D3 on neoplastic transformation induced by 60Co gamma-rays or 252Cf fission neutrons in Balb/c 3T3 cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] and okadaic acid were individually examined on the neoplastic transformation of Balb/c 3T3 cells which were exposed to either 60Co gamma-rays or 252Cf fission neutrons. The addition of 1 alpha,25(OH)2D3, or TPA, enhanced the transformation induced by gamma-rays or low doses of fission neutrons. No enhancement was observed by the addition of okadaic acid except at toxic concentrations (5 ng/ml) and with higher doses of radiation. Moreover, the enhancement of transformation by either 1 alpha,25(OH)2D3 or TPA decreased as the radiation dose was increased. The enhancement ratio, calculated by least-square analysis from 0 Gy to 1 Gy, was greater for 1 alpha,25(OH)2D3 than for TPA, and also greater for gamma-ray irradiation than for neutron irradiation. These results suggest that the promotion of radiation-induced transformation depends on the level of the initial damage caused by radiation; and that the differences in the enhancement properties of different chemicals may be due to different individual triggering mechanisms involved in the transformation process.

3T3 Cells

[Immunohistochemical studies on S-100 beta positive structures in the human hippocampus in regard to age and morphological change of dementia].

The histological localization of S-100 beta protein in the hippocampus of human autopsy brains of 47 males (71-103 years old) and 90 females (56-104 years old) was studied immunohistochemically. Astrocytes and their processes were positively stained, but neuronal cells were not stained. However, Alzheimer's neurofibrillary tangle-like, senile plaque-like and fibrillary spindle figures were stained positively. S-100 beta positive structures increased in grade with age, but not always equally on Alzheimer's neurofibrillary tangles or senile plaques stained by Bodian method. Astrocytes decreased in number with age, and showed marked compensatory hypertrophy of their processes. S-100 beta positive structures seemed to be related to astroglial changes in terms of degeneration or loss of synapses.

Aged

[The first case of infantile type neuronal ceroid-lipofuscinosis in Japan].

We reported the first case of typical infantile-type neuronal ceroid-lipofuscinosis (INCL) in Japan. The patient was a 1-year-old girl presenting with rapidly progressive psychomotor deterioration and blindness. Muscular hypotonia, microcephaly and myoclonic jerks became marked with the progression of her disease. Diminution in amplitude of EEG, VEP and ERG was prominent in the initial stage, but ABR was normal. MRI showed progressive brain atrophy. Electron microscopic examination of the biopsied skin revealed granular matrix, the specific inclusion bodies, in the epithelial cell of sweet glands. Many sea-blue histiocytes were demonstrated in her bone marrow samples. INCL is a common progressive encephalopathy in the Scandinavian countries, but a typical case had not yet been fully reported in Japan. This prompted us to report our case. Future reports are need for the study of INCL in Japan.

Biopsy