Fetal gender prediction based on maternal plasma testosterone and insulin-like peptide 3 concentrations at midgestation and late gestation in cattle.

We compared maternal plasma testosterone and insulin-like peptide 3 (INSL3) concentrations between dams carrying a male versus female fetus from early to late gestation and examined the application of maternal hormonal concentrations to fetal gender prediction in dairy and beef cattle. Blood samples were collected from Holstein cows or heifers (N = 31) and Japanese Black beef cows (N = 33) at 1-month intervals at 2 to 8 months of gestation. Fetal gender was confirmed by visual observation of external genitalia of calves just after birth. Plasma testosterone and INSL3 concentrations were determined by enzyme-immunoassay. Fetal genders were judged based on cutoff values of maternal testosterone and INSL3 concentrations (male, if it was ≥ cutoff value; female, if < cutoff value), which we set for each hormone at each gestational month using receiver operating characteristic curves. Plasma testosterone concentrations were higher for dams with a male fetus than those with a female at 4, 5, 7, and 8 months for the dairy cattle (P < 0.05) and at 4, 5, 6, and 8 months for the beef cows (P < 0.05). Plasma INSL3 concentrations were higher for dams with a male fetus than those with a female at 2 and 6 months for the dairy cattle (P < 0.05) and at 4 to 8 months for the beef cows (P < 0.05). The predictive values and detection rates for fetal gender prediction based on maternal testosterone concentrations were 75.8% to 79.3% for dairy cattle at 5 and 7 months and for beef cows at 5 and 6 months, whereas those values by maternal INSL3 concentrations were 71.0% to 72.4% for the dairy cattle at 6 months and beef cows at 4 and 8 months. When multiple time points of testosterone and INSL3 concentrations at several midgestation and late gestation months were considered for fetal gender prediction, predictive values were 89.3% (5-7 months) and 85.7% to 88.0% (4-6, 8 months) for the dairy and beef breeds, respectively. Maternal testosterone and INSL3 concentrations in dams carrying a male fetus were higher than those carrying a female at midgestation and/or late gestation in Holstein and Japanese Black beef cattle. Nearly, 80% accuracy was obtained for fetal gender prediction by a single time point of maternal plasma testosterone concentrations at midgestation. Nearly 90% accuracy for the prediction was obtained when multiple time points of testosterone and INSL3 concentrations from midgestation to late gestation were considered.

Population research of genetic polymorphism at amino acid position 631 in chicken Mx protein with differential antiviral activity.

A single amino acid substitution between Asn and Ser at position 631 in the chicken Mx protein has been reported to determine resistant and sensitive antiviral activity. In this study, we investigate whether various kinds of chicken breeds and jungle fowls carry the resistant or sensitive Mx allelic gene by using the mismatched PCR-restriction fragment length polymorphism (RFLP) technique. In total, 271 samples from 36 strains of 17 chicken breeds and from 3 kinds of jungle fowls were examined. The rates of the resistant Mx gene and sensitive gene were 59.2% and 40.8%, respectively. Only a Red jungle fowl captured in Laos carried the resistant Mx gene, and the other three Red jungle fowls from Indonesia and Gray and Green jungle fowls all had the sensitive Mx gene. These results were confirmed by the determination of amino acid sequences in the GTPase effector domain of jungle fowls.

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice.

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.

X-irradiation induces up-regulation of ATM gene expression in wild-type lymphoblastoid cell lines, but not in their heterozygous or homozygous ataxia-telangiectasia counterparts.

Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradiated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM + / + ), AT heterozygotes (ATM + / - ), or AT homozygotes (ATM - / - ), respectively. In ATM + / + LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this time. The results obtained with the ATM + / - LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM + / - LCL. Twelve of the mutations in the ATM - / - LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM - / - LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.

Arrest of human dendritic cells at the CD34-/CD4+/HLA-DR+ stage in the bone marrow of NOD/SCID-human chimeric mice.

Human dendritic cell (DC) precursors were engrafted and maintained in NOD/SCID- human chimeric mice (NOD/SCID-hu mice) implanted with human cord blood mononuclear cells, although no mature human DCs were detected in lymphoid organs of the mice. Two months after implantation, bone marrow (BM) cells of NOD/SCID-hu mice formed colonies showing DC morphology and expressing CD1a in methylcellulose culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The CD34-/CD4+/HLA-DR+ cell fraction in NOD/SCID-hu mouse BM generated CD1a(+) cells that were highly stimulatory in mixed leukocyte reactions in culture with GM-CSF and TNF-alpha. These results suggest a strong potential for NOD/SCID-hu BM to generate human DCs, although DC differentiation may be blocked at the CD34-/CD4+/HLA-DR+ stage. (Blood. 2001;97:3655-3657)

X-rays induce dose-dependent and cell cycle-independent accumulation of p21(sdi1/WAF1).

Cell cycle arrest at the G1 checkpoint is governed by a function of wild-type p53. We assessed the behavior of the sdi1 gene, which codes for a 21kDa potent inhibitor of cdk/cyclins, after X-irradiation. X-irradiation induced sdi1 mRNA accumulation and G1 arrest only in cells possessing wild-type p53. Elevation of p21(sdi1/WAF1) was preceded by p53 accumulation, which occurred despite p53 mRNA constancy in normal cells growing in the log phase. The quantity of accumulated p53 and p21(sdi1/WAF1) was radiation dose dependent. A decrease in the S phase cell population in normal cells observed after irradiation reached a minimum at less-than-maximum levels of p53 and p21(sdi1/WAF1). Furthermore, an accumulation of p53 and p21(sdi1/WAF1) was also observed when cells were synchronized in the G0, G1 and S phase and X-irradiated. These results indicated that an X-ray induced p53 and p21(sdi1/WAF1) accumulation mechanism exists throughout the cell cycle, and that the signal strength induced by X-irradiation is dose-dependent.

NK-mediated elimination of mutant lymphocytes that have lost expression of MHC class I molecules.

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.

Preferential induction of RET/PTC1 rearrangement by X-ray irradiation.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RET/PTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RET/PTC1, -2 and -3 are known to be the three major forms. High frequencies of RET/PTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RET/PTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RET/PTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RET/PTC3 was detected only 7 days after X-irradiation, and no transcript of RET/PTC2 was detected. These results are supported by the results of an in vitro study. The RET/PTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RET/PTC3 was induced at a much lower frequency, and no induction of RET/PTC2 was observed. These results suggest that the preferential induction of the RET/PTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

Decreased proportion of CD4 T cells in the blood of atomic bomb survivors with myocardial infarction.

Epidemiological studies of the atomic bomb survivors have suggested dose-related increases in mortality from diseases other than cancer. Cardiovascular disease is one such noncancer disease for which increases in both mortality and incidence have been found to be associated with radiation dose. Immunological studies have revealed long-term impairment of T-cell-mediated immunity, especially involving deficiencies of CD4 helper T cells, in atomic bomb survivors. In the present study, we investigated whether decreases in CD4 T cells were associated with myocardial infarction in atomic bomb survivors. Of 1,006 survivors examined to determine the proportion of CD4 T cells in peripheral blood lymphocytes, 18 persons had a history of myocardial infarction. The proportion of CD4 T cells was significantly decreased with increased radiation dose [corrected]. Further, the prevalence of myocardial infarction was significantly greater in individuals with a lower proportion of CD4 T cells. These results suggest that myocardial infarction in atomic bomb survivors may be associated with defects in CD4 helper T cells.

Mutant p53: epigenetic mutator of the T-cell receptor via induction of methylation.

The mechanism and effects of epigenetic alterations in human carcinogenesis are not well understood, except that cancers often have alterations in the methylation status of their genomes. Additionally, human cancers, including aggressive T-cell leukemias and lymphomas, have a high frequency of p53 mutations, particularly missense mutations, which raises the possibility of gain-of-new-function proteins, but the new proteins' oncogenic functions are mechanistically ill-defined. To investigate the mechanisms behind the high prevalence of p53 tumor suppressor gene mutations in aggressive or relapsed T-cell leukemias, we transfected Jurkat cells null for p53 protein with a temperature-sensitive p53 mutant. We showed that this mutant p53 abrogated expression of the T-cell antigen receptor (TCR) by affecting the methylation of an at least 20-kb region of DNA, 5'to the TCR beta-chain gene enhancer region, which includes TCRbetaC1 and betaC2. Expression of the TCR is restored when the temperature is reduced to 32 degrees C, at which temperature the mutant p53 regains wild-type function. The TCR, a common site of dysfunction in T-cell malignancies, is the principal signal transduction moiety controlling both T-cell activation and activation-induced apoptosis. These results suggest a new role for mutant p53-as an epigenetic mutator, bridging p53, methylation, and transcriptional silencing-and suggest novel mechanisms in immunosuppression and cancer progression.

p53 mutations in tumor and non-tumor tissues of thorotrast recipients: a model for cellular selection during radiation carcinogenesis in the liver.

Concerns over cancer development from exposure to environmental sources of densely ionizing, high linear energy transfer (LET) radiation, such as alpha-particles from radon, is a current public health issue. The study of tumors attributable to high LET irradiation would greatly augment our insights into the biological mechanisms of carcinogenesis. Chronic low-dose-rate internal exposure to alpha-radiation from thorium dioxide deposits following intravascular administration of the radiographic contrast agent Thorotrast is known to markedly increase the risk of cancer development, especially that of hepatic angiosarcomas and cholangiocarcinomas. Although the mechanism is hypothesized to be via cellular damage, DNA being a major target, wrought by the high LET alpha-particles, the specific genes and the actual sequence of events involved in the process of transforming a normal cell into a malignant one are largely unknown. To shed some light on the molecular mechanisms of cancer development during a lifetime exposure to alpha-radiation, we analyzed the most commonly affected tumor suppressor gene in humans, p53, in 20 Thorotrast recipients who developed cancer, mostly of hepatic bile duct and blood vessel origin. Of the 20 cases, 19 were found to harbor p53 point mutations. Moreover, the accompanying non-tumor tissues from these patients also had p53 mutations, albeit at lower frequency. The distribution pattern of the point mutations was significantly different between the non-tumor and tumor tissues, with most mutations in malignant tissues located in the highly conserved domains of the p53 gene. Our results support the idea that p53 mutations are important in the genesis of Thorotrast-induced tumors but that these point mutations are a secondary outcome of genomic instability induced by the irradiation. Additionally, non-tumor cells harboring p53 mutations may gain some survival advantage in situ but mutations in the domains responsible for the formation of structural elements critical in binding DNA may be necessary for a cell to reach full malignancy.

Close correlation between a p53 or hMSH2 gene mutation in the tumor and survival of hepatocellular carcinoma patients.

We analyzed 42 hepatocellular carcinomas (HCC) (38 patients) for mutations in the DNA mismatch repair gene hMSH2 and the p53 tumor suppressor gene, a possible upregulater of hMSH2. Mutations of the hMSH2 or p53 gene were detected in 13 patients (34%). There were no patients who possessed mutations in both genes. There was a significant correlation between mutation of either gene and pathological indicators of malignancy. The survival rate of patients with hMSH2 or p53 gene mutation-positive tumors was much poorer than that with hMSH2 and p53 gene mutation-negative tumors (p=0.0012). Our results suggest that mutations in the p53 or hMSH2 gene may closely correlate with the survival of hepatocellular carcinoma patients.

In vivo somatic mutations in Werner's syndrome.

The frequencies of mutant erythrocytes with loss of heterozygosity at the glycophorin A (GPA) locus and mutant CD4+ T cells lacking surface expression of the T-cell receptor alphabeta (TCR)/CD3 complex were measured by flow cytometry for Japanese Werner's syndrome (WRN) patients. The hemizygous and homozygous GPA mutant frequencies (GPA Mfs) and the TCR/CD3-defective mutant frequency (TCR Mf) in WRN patients were found to be significantly higher than those in normal controls in the same age range. However, because these Mfs in the patients are only about twice those in controls, it is difficult to conclude that the WRN gene mutations cause instability of somatic genes. This contrasts markedly with Bloom's syndrome (BLM) patients, whose GPA and TCR Mfs were previously reported to increase about 50- and 15-fold, respectively. The difference in Mfs is one aspect of the large variation in the phenotype observed between WRN and BLM patients, suggesting a different role of the responsible genes, both of which belong to the RecQ DNA helicase gene family, in the control of somatic mutagenesis.

Lifespan of human memory T-cells in the absence of T-cell receptor expression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

Flow cytometry measurements of subsets of T, B and NK cells in peripheral blood lymphocytes of atomic bomb survivors.

Previous studies of blood cells from atomic bomb survivors have shown that frequencies of chromosome aberrations and somatic mutations are elevated in heavily exposed survivors and that T-cell functions and the number of mature T cells are decreased in the survivors who were exposed to radiation as adults. Current progress in flow cytometry allows a sophisticated analysis of various subsets of T, B and NK cells. In the present study, proportions of such subsets in peripheral blood lymphocytes from atomic bomb survivors (159 survivors estimated to be exposed to > or =1.5 Gy) and 252 controls were measured using multiple combinations of monoclonal antibodies to lymphocyte differentiation antigens to investigate whether the previous radiation exposure had altered the composition of the subsets. Among T-cell subsets, the proportion of CD4+ T-cell subsets was decreased significantly in the heavily exposed survivors; this tendency was apparent for the CD4+CD45RA+ naive T-cell subset. However, there were no significant differences in the proportions of CD8+ T-cell subsets between the exposed survivors and controls. As for the B-cell subsets, the proportion of both CD5+ and CD5 B cells as well as CD23+ and CD23- B cells increased in the heavily exposed survivors. Further, no effect of radiation was found in the proportion of NK-cell subsets. These results strongly suggest that previous radiation exposure altered the composition of T and B cells in the peripheral blood of atomic bomb survivors, and they raise the possibility that atomic bomb radiation may have affected the developmental processes of T and B cells.

Oral administration of tritiated water (HTO) in mouse. III: Low dose-rate irradiation and threshold dose-rate for radiation risk.

PURPOSE: To investigate the biological effect of tritium on mouse at low dose-rates. MATERIALS AND METHODS: Mice ([C57BL/6N x C3H/He]F1) were exposed to beta-rays by continuous administration of tritiated drinking water throughout their lives at low dose-rates of 3.6, 0.9, and 0.2 mG/day. RESULTS: Including the previous study, the tumour frequency was 70 to approximately 80% for exposure in the range 240 mGy/day to 9.6 mGy/day. Frequency of tumours decreased with decrease of dose-rate to 50% comparable to the controls. Restricting to thymic lymphomas, a linear relationship in a semi-log plot was found between the frequency and the dose-rate above a threshold dose-rate of 12 mGy/day. There was a 'tail' to this relationship down to 0.9 mGy/day. A similar pattern resulted for the relationship between the life-shortening and the dose-rate. The threshold dose-rate of 3H beta-rays, 2 mGy/day (with a tail down to 0.2 mGy/day), was much lower than that of gamma-rays, 20 mGy/day (tail down to 2 mGy/day) derived from other studies. CONCLUSIONS: These studies suggest that there exists the threshold dose-rate in the biological effects of radiation, and that the threshold dose-rate for 60Co gamma-irradiation is higher than that for 3H beta-irradiation.

Multiple, unique, and common p53 mutations in a thorotrast recipient with four primary cancers.

Four primary cancers found at autopsy of a patient who received the thorium-based contrast agent Thorotrast 50 years ago and who was healthy up until a few months before his death from liver failure were analyzed for p53 mutations. The data suggest that the chronic alpha-irradiation may be a large causative factor. Multiple mutations were found in all the cancer tissues: two foci of a cholangiocellular carcinoma, a tubular adenocarcinoma of the stomach, a squamous cell carcinoma of the lung, and an adenocarcinoma of Vater's ampulla. The total number of point mutations detected were 13. Moreover, homozygous aberrations were detected in a large area of normal small intestine and noncancer liver tissues suggesting that nontumor cells which harbored p53 abnormalities gained a survival advantage and clonally expanded.

Radiation sensitivity of human hair follicles in SCID-hu mice.

We developed an experimental model for studying the growth and epilation of the human hair follicle by implanting human scalp tissue onto immunodeficient C.B-17 scid/scid mice. The skin grafts showed continuous growth of black human hairs for at least 1 year and maintained the normal histological structure of a human hair follicle and other tissues associated with the skin. Using this in vivo model, we evaluated the effect of irradiation on the function of human hair follicles. Localized X irradiation (1 to 6 Gy) induced hair loss dose-dependently and synchronously in the third week after irradiation. The hairs undergoing epilation showed a gradual decrease in width toward the root. The minimum width at the thinnest portion of the surviving hair 4 weeks after irradiation suggested that epilation resulted from the breaking of hairs when the hair width decreased to less than 20 microm. After the highest-dose irradiation, the normal structure of the hair bulb was totally abrogated, and long and narrow epithelial tissues associated with regressed papillary cells remained. The surviving epithelia were morphologically similar to the outer epithelial sheath of the follicle associated with palisadic basal cell layers. In the third week some cells in the basal layers of the surviving epithelium in each follicle expressed proliferating cell nuclear antigen. By about 9 weeks after irradiation, the complete structure of the follicle regenerated, with hair growth activity even in the grafts irradiated at the highest dose, although about 30% of the hairs did not regrow. These findings suggest that follicular stem cells that survive high-dose exposure in the sheath-like epithelial tissue can reproduce the complete follicle structure. This animal model can be used to assess the effects of radiation exposure on human skin and to identify and characterize human follicular stem cells.