Prenatal radiation-induced limb defects mediated by Trp53-dependent apoptosis in mice.

We reported previously that in utero radiation-induced apoptosis in the predigital regions of embryonic limb buds was responsible for digital defects in mice. To investigate the possible involvement of the Trp53 gene, the present study was conducted using embryonic C57BL/6J mice with different Trp53 status. Susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both Trp53 status and the radiation dose; i.e., Trp53 wild-type (Trp53(+/+)) mice appeared to be the most sensitive, Trp53 heterozygous (Trp53(+/-)) mice were intermediate, and Trp53 knockout (Trp53(-/-)) mice were the most resistant. These results indicate that induction of apoptosis and digital defects by prenatal irradiation in the later period of organogenesis are mediated by the Trp53 gene. These findings suggest that the wild-type Trp53 gene may be an intrinsic genetic susceptibility factor that is responsible for certain congenital defects induced by prenatal irradiation.

Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status.

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.

Quantitative analysis of bowel gas using plain abdominal radiograph in patients with irritable bowel syndrome.

OBJECTIVE: Ideally, the diagnosis of irritable bowel syndrome (IBS) would be achieved using a minimal number of procedures. It is presumed that bowel gas is related to IBS, and it is easily visualized by plain abdominal radiograph. In the present study, to clarify the relationship between IBS and the quantity of bowel gas, the measured bowel gas volume using plain abdominal radiographs was compared with the pathology of IBS. METHODS: Plain abdominal radiographs were digitized and transmitted to a computer (computed radiography) in 30 IBS patients and 30 normal controls. The quantity of bowel gas, determined as the pixel value on images and standardized by physique, was defined as the gas volume score (GVS). Using the mean +/- 2SD of GVS in the control group as the normal score, IBS patients were divided into three groups: high, normal, and low. To examine the sequential reproducibility of a similar quantity of bowel gas, a second plain abdominal radiography was performed about 2 months later, and the GVS were compared. The colonic transit time was determined using radiopaque markers. RESULTS: There was a strong correlation between the quantities of bowel gas measured by two independent gastroenterologists. The mean GVS of IBS patients was significantly higher than that in the control group (p < 0.001). The sequential reproducibility was recognized in all 10 IBS patients. There was no significance between colonic transit time and GVS, nor between symptoms and GVS. CONCLUSIONS: Abdominal gas was analyzed objectively by using GVS, and GVS was considered to represent a useful tool for the diagnosis of IBS.

UVC-induced apoptosis in human epithelial tumor A431 cells: sequence of apoptotic changes and involvement of caspase (-8 and -3) cascade.

Human epidermoid tumor A431 cells underwent apoptosis following exposure to ultraviolet C (UVC). The apoptosis was of the interphase death type, and mostly occurred within one cell cycle, independent of the cell-cycle phases. We further examined the detailed sequential order of apoptotic changes in cells after UVC exposure and the involvement of caspases using six caspase inhibitors. The loss of mitochondrial transmembrane potential (delta psi m) appeared in the earliest phase; subsequently, the chromatin condensation and DNA-fragmentation occurred. Cell shrinkage and loss of the plasma-membrane integrity, judged by propidium iodide (PI) staining, were observed in the later phase. A broad-spectrum caspase inhibitor, z-VAD-fmk, completely prevented all apoptotic changes, except for the depletion of delta psi m. Both Ac-DEVD-CHO and Ac-IETD-CHO, inhibitors of caspase -3 and -8, respectively, effectively inhibited typical chromatin condensation to almost the same extent. However, the nuclei still showed partial condensation. A caspase -9 inhibitor, Ac-LEHD-CHO, did not prevent chromatin condensation, though it partially inhibited cell-size reduction and PI-stainability. None of the caspase inhibitors could inhibit the delta psi m reduction. These results strongly suggest that the collapse of delta psi m is not a part of the central apoptotic machinery, and that caspase cascade(s), especially caspase-8 to -3, play an important role in UVC-induced apoptosis in A431.

Induction of apoptosis by beta radiation from tritium compounds in mouse embryonic brain cells.

Induction of apoptosis by tritium exposure was investigated in both cultured embryonic mid brain cells and brain sections of embryos and of newborns in mice. In the cultures of mid brain cells, addition of methyl-3H-thymidine (3H-TdR) (21 kBq mL(-1)) and tritiated water (5.616 MBq mL(-1)) induced late appearances and low percentages of apoptosis when compared to x-irradiation at the ID50 dose, the inhibitory dose that reduced cellular differentiation by 50% of the control. A significant increase in p53 protein was detected about 2 h before the marked appearance of apoptosis. The pregnant mice were given an intraperitoneal injection of tritiated water at the concentration of 481.8 kBq g(-1) of body weight on gestation day 12.5, by which treatment behavioral changes in the offspring occurred. Increased apoptotic cells were observed in the neural tube of embryos from 1 d after the injection to 1 wk postnatal age. Apoptosis induced by x-rays appeared 2 h after irradiation, with a peak at 4 h. Increase of apoptotic cells was also found in the brain cortexes of newborns. The percentage of apoptosis in the brain was higher in the prenatal tritiated water exposed mice than in the prenatal x-irradiated mice. Possible mechanisms on apoptosis and its relation to the higher relative biological effectiveness value of tritium beta-rays are discussed.

Radiation-induced apoptosis and limb teratogenesis in embryonic mice.

In utero irradiation of the fetus during the period of organogenesis induces a dramatic increase in malformation. However, the mechanisms underlying the teratogenesis remain to be elucidated. In the present study, the correlation between radiation-induced apoptosis and limb malformation was examined in mice. The mice were exposed to X rays in utero on day 11 of gestation during the period of organogenesis of limb buds. A marked increase in the number of apoptotic cells in the predigital regions in the forelimb buds was detected 4 h after irradiation. The preinterdigital regions of the forelimb buds did not show such an increase at the same time. Aphlangy and ectrodactyly were the main types of anomalies observed on day 19 in the limbs of the fetuses irradiated with 5 Gy. The increases in prenatal death and teratogenesis in limb digits in living fetuses were dependent on dose. The possible mechanisms involved are discussed.

Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction.

PURPOSE: To develop and validate non-fluorescent chromosome painting for bright-field microscopy using the peroxidase/diaminobenzidine (DAB) reaction. MATERIALS AND METHODS: Peripheral blood lymphocytes were taken from patients with uterine cancer who had received heavy-ion radiation therapy. Chromosome slides were treated with RNase and pepsin, denatured mildly, hybridized with a biotinylated DNA probe specific for whole-chromosome 4 and stained using the peroxidase/DAB reaction with an avidin-biotin amplification. The slides were analysed under a bright-field microscope and an atomic force microscope. The detection rate of chromosome aberrations by DAB painting was compared with that obtained by dual analysis of Giemsa staining and FISH painting. RESULTS: When chromosomes 4 were painted, 11.5% of unstable aberrations were detected by DAB painting, while 10.8% of them were found by dual analysis of Giemsa staining and FISH painting. CONCLUSION: A DAB painting method that can effectively detect rearranged aberrations was established. It has advantages over FISH painting: the preparations can be analysed by bright-field microscope, can be preserved permanently and are suitable for analysis by an automated system.

Adaptive response in embryogenesis: I. Dose and timing of radiation for reduction of prenatal death and congenital malformation during the late period of organogenesis.

An adaptive response was demonstrated during embryogenesis in mice. Whole-body irradiation at a dose of 0-50 cGy was given to condition pregnant ICR mice on day 9 to day 11 of gestation. Then their whole bodies were exposed to a challenging dose of 5 Gy on the next day. The numbers of living fetuses, prenatal deaths and living fetuses with external gross malformations were determined on day 19. A conditioning dose of 30 cGy on day 11 significantly increased the rate of living fetuses and reduced the incidence of congenital malformations induced by a 5-Gy dose on day 12. This indicates the existence of a critical dose and timing for administering a conditioning dose for radioadaptation during the late period of organogenesis in mice. The possible mechanisms involved are discussed.

Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

Patient registration and treatment allocation in multicenter clinical trials using a FAX-OCR system.

This article describes the design and results of implementation of an automated patient registration and treatment allocation system (RETAS) used in multicenter clinical trials. RETAS was developed using a FAX-OCR system by which handwritten Japanese and English characters, as well as numericals and forms with check boxes, are sent from participating institutions by Fax, processed using an optical character reader, and then transmitted to a host computer at a statistical center. Based on the facsimile data, RETAS can automatically review eligibility, collect patient identification data and provide a randomized treatment allocation. RETAS permits uninterrupted, unattended operation at a statistical center, 24 hours a day, 7 days a week. Therefore, it drastically decreases the workload of personnel at the statistical center needed to support central telephone registration coverage. Consequently, staff members are free to focus on patient registration, treatment allocation, and follow-up of patients. The treatment allocation procedure in this system is based on Pocock and Simon's minimization method combined with Zelen's method for institution balancing. By this system it was possible to balance treatment numbers for each level of various prognostic factors over an entire trial and, at the same time, balance the allocation of treatments within an institution. The system currently supports the protocol of a clinical trial for Adjuvant Chemo-Endocrine Therapy for Breast Cancer in West Japan.

The study of tumor necrosis factor beta gene polymorphism in lung cancer patients.

BACKGROUND: In recent years numerous reports have discussed the relationship between the human leukocyte antigen and lung cancer. However, the genetic background of lung cancer has not yet been precisely clarified. METHODS: To investigate the genetic background of lung cancer, the human leukocyte antigens in 159 normal healthy control subjects and 102 lung cancer patients were studied, and restriction fragment length polymorphism analysis of the tumor necrosis factor (TNF) beta gene in 165 normal healthy control subjects and 135 lung cancer patients was performed. RESULTS: Lung cancer patients showed a high frequency of human leukocyte antigen B61; however, no statistical difference was found. In the lung cancer patients, the TNF beta 10.5/10.5-kb allele was found at a low frequency, 38.5%, compared to 53.3% in normal controls (chi 2 = 7.51, P = 0.011, corrected P = 0.033, relative risk = 0.77). In the relationship between the histologic types and the TNF beta gene, the TNF beta 10.5/10.5-kb allele showed low frequencies: 38.5% in adenocarcinoma, 38.2% in squamous cell carcinoma, and 27.8% in small cell carcinoma, although no statistical difference was shown. In relation to the postoperative survival period, the TNF beta 10.5/10.5-kb allele was associated with prolonged survival. CONCLUSIONS: The TNF beta 10.5/10.5-kb allele may be associated with resistance to lung cancer and with a better prognosis.

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.

Clinical features of the differentiated and undifferentiated types of advanced gastric carcinoma: univariate and multivariate analyses.

This study was done to define clinical features for the different pathological types of advanced gastric carcinoma. One thousand one hundred three patients were identified and classified into two groups: 479 patients (43.4%) had a differentiated adenocarcinoma and 624 patients (56.6%) had an undifferentiated adenocarcinoma. Patients with the undifferentiated type were more likely to have large invasive tumors and a higher incidence of peritoneal dissemination. Conversely, the patients with the differentiated type were more likely to have a liver metastasis. Multivariate analysis, using Cox' proportional hazard model adjusted for sex, age, and other factors, suggested that tumor size was one of the seven most independent prognostic factors in patients with the undifferentiated type (relative risk = 1.01), but this parameter lost prognostic value in patients with the differentiated type. With regard to correlation between survival time and tumor size, the larger the tumor (over 10 cm), the shorter the survival time of patients with the undifferentiated type, as compared to findings in patients with the differentiated type (P less than 0.01). Thus, differences in clinical characteristics, including characteristics in the individual patients, extent of tumor, distant metastasis, prognostic factors, and prognosis correlate with the histopathological type of gastric carcinoma.

Death due to recurrence following curative resection of early gastric cancer depends on age of the patient.

This study was done to define the relationship between age at the time of surgery and the prognosis after curative resection for patients with an early gastric cancer. Three hundred and eighty-two patients were identified and 25 patients died of tumour recurrence. Overall, the cumulative survival rate was 94.9% at 5 years and 92.4% at 10 years. Patients with a recurrence of the gastric cancer tended to be older, were more likely to have large differentiated type of tumour and lymph node metastases were often present. Stratified into age-classified groups, the survival rate decreased with increase of age (for patients under age 34 years, 35 to 44, 45 to 54, 55 to 64, 65 to 74, over age 75 years, the 5-year survival rates were 100.0, 97.7, 97.6, 94.2, 94.1 and 84.4 (%]. Of the 25 patients with a tumour recurrence and who died, the survival time of 18 patients over age 55 years was significantly shorter than that of seven patients under age 54 years (median, 1.7 vs 5.6 years, P less than 0.05). The multivariate analysis showed that, over and above the differentiated type of tumour (P less than 0.01) and the presence of lymph node metastases (P less than 0.01), age was one of the prognostic factors (P less than 0.05). We conclude that age at the time of primary surgery is a significant factor in patients with an early gastric cancer.

Biochemical and immunological characterization of murine leukemia viruses that are paralysis-inducing in rats.

The molecular size and pI of the viral structural proteins of four PVC viruses (PVC111, PVC211, PVC321 and PVC441) were compared by single or two-dimensional polyacrylamide gel electrophoresis. PVC111 had slightly larger p15E and gPr85 molecules (about 0.5 kilodalton) than did the other PVC viruses. On the other hand, the virion structural proteins p30, p15, p12E and p12 from all the viruses had the same molecular sizes and pIs. The gp70s and p10s from all the viruses showed the same molecular sizes. A monoclonal antibody to gp70 of PVC321 virus recognized the gp70s of all PVC viruses, but not the gp70s of four clones of the wild mouse ecotropic viruses, Friend murine leukemia viruses (F-MuLV), AKR ecotropic MuLV, dual-tropic F-MuLV or NZB endogenous xenotropic MuLV, revealing that these four PVC viruses are homologous with each other, but distinct from the known mouse retroviruses.

Intraarticular epiphyseal osteoid osteoma of the distal femur.

A 9-year-old boy had pain in the medial side of the right knee with limited range of motion and limping. Roentgenography showed a small sclerotic shadow (8 X 8 mm2) in the medial femoral condyle, where bone scintigraphy revealed a high uptake area and angiography showed a nidus. Five months after initial presentation, en bloc excision was done through the posteromedial approach. Histological examination showed a network of osteoid trabeculae, differentiated osteoblasts, and multinucleated giant cells in this nidus, which were compatible with those of osteoid osteoma. Complete relief of pain was obtained at follow-up 1 year after the operation.

[Tissue reaction of the bone marrow to glass ceramic powder in the femurs of rats fed on a low-calcium diet].

Glass ceramic powder was injected into the bone marrow of 36 rats which were fed a low calcium (21 rats) or normal (15 rats) diet. They were sacrificed 7 or 8, 32 and 60 days after treatment. Histomorphometrical measurement revealed that the ratio of length of bone tissue which was in contact with glass ceramic mass to the circumference of each mass was larger in 32-day and 60-day groups than in the 7 or 8-day groups. Osteocyte-like cells were observed in the narrow space between ceramics and bone tissue in all specimens. Giant cells were seen in 1-2% of the glass ceramic surfaces in low calcium diet group and in 0.3% in normal diet group. No inflammatory reaction such as aggregation of lymphocytes nor leukocyte infiltration was observed in any specimens. Eighty-two percent of the surface of glass ceramic mass was covered with bone tissue 60 days after treatment in normal diet group.

Non-A, non-B hepatitis related AN6520 Ag is a normal cellular protein mainly expressed in liver. II.

Detection of AN6520 Ag/Ab in human sera had indicated a close association with non-A, non-B hepatitis (NANBH). In this study, we investigated the immunochemical nature of AN6520 Ag and measured the amounts in various human and chimpanzee organs in order to clarify the association with NANBH. AN6520 Ag was found to be composed of polypeptide(s) with an apparent molecular weight of 45,000 daltons (45 kD), which are noncovalently linked together. Human antibodies in convalescent sera from NANBH patients as well as monoclonal antibodies were found to recognize only the high-order structure of the antigen, whereas rabbit antibody recognized both the high-order structure and the reduced form of 45 kD polypeptide(s). AN6520 Ag could be detected in most of the livers tested including those without any liver damage and fetal livers; their amounts varied considerably from each other. The antigen could be detected also in organs other than liver, but in contrast to liver, the amounts were small and did not vary as much between individuals. From the data of immunoblotting using rabbit antibody, our observed variation of antigen content in liver was considered to be due to the difference in expression of 45 kD polypeptide(s). Although no specific relationship was found between the amount of the antigen in liver and NANBH, the antigen was found to increase several times in livers of chimpanzees after the inoculation of NANBH virus. These data suggest that AN6520 Ag is a normal cellular protein existing mainly in liver and that its quantity may vary under some conditions such as NANBH.

Relationship between the cellular resistance to Friend murine leukemia virus infection and the expression of murine leukemia virus-gp70-related glycoprotein on cell surface of BALB/c-Fv-4wr mice.

Fv-4r is a dominant resistance gene which controls resistance of mice to exogenous infection with ecotropic murine leukemia viruses. Cell lines with various degrees of resistance to ecotropic Friend murine leukemia virus infection were established from BALB/c-Fv-4wr mice which are partially congenic with BALB/c. The degree of resistance of these cell lines correlated well to the amount of glycoprotein with mol wt 80 K on cell surface. The resistance of cells was reduced by the treatment of glycosylation inhibitors, tunicamycin and 2-deoxy-D-glucose. The results indicate that the Fv-4 resistance is ascribed to the unique glycoprotein on cell surface.

A culture technique for chromosome analysis in human myeloid leukemias.

A simple culture technique for the study of cells from myeloid leukemias and other blood disorders of myeloid series is described. The procedure is the same with that of the ordinary cultures, except for the addition of colony stimulating factor. A large number of mitotic cells can be obtained, and they are quite suitable for banding treatments. Clear banding patterns are consistently obtained.