NATURAL RADIOACTIVITY LEVEL AND ELEMENTAL COMPOSITION OF SOIL SAMPLES FROM A HIGH BACKGROUND RADIATION AREA ON EASTERN COAST OF INDIA (ODISHA).

A comprehensive study was carried out to determine the radioactivity concentration of soil samples from different sites of a high background radiation area in the eastern coast of India, Odisha state. The dose rate measured in situ varied from 0.25 to 1.2 µSv h-1 The gamma spectrometry measurements indicated Th series elements as the main contributors to the enhanced level of radiation and allowed the authors to find the mean level of the activity concentration (±SD) for 226Ra, 228Th and 40K as 130±97, 1110±890 and 360±140 Bq kg-1, respectively. Human exposure from radionuclides occurring outdoor was estimated based on the effective dose rate, which ranged from 0.14±0.02 to 2.15±0.26 mSv and was higher than the UNSCEAR annual worldwide average value 0.07 mSv. Additionally, X-ray fluorescence analysis provided information about the content of major elements in samples and indicated the significant amount of Ti (7.4±4.9 %) in soils.

Comparative dosimetry for radon and thoron in high background radiation areas in China.

The present study focuses on internal exposure caused by the inhalation of radon and thoron progenies because the internal exposures have not yet been clarified. For their dose assessment, radon, thoron and thoron progeny concentrations were measured by passive monitors over a long period (for 6 months). Consequently, radon, thoron and equilibrium equivalent thoron concentrations were given as 124 ± 78, 1247 ± 1189 and 7.8 ± 9.1 Bq m(-3), respectively. Annual effective doses are estimated to be 3.1 ± 2.0 mSv for radon and 2.2 ± 2.5 mSv for thoron. Total dose are estimated to be 5.3 ± 3.5 mSv a(-1). The present study has revealed that the radon dose was comparable with the thoron dose, and the total dose was ∼2 times higher than the worldwide average.

Development of a tool system to support endoscope.

Natural Orifice Translumenal Endoscopic Surgery (NOTES) is a type of minimally invasive surgery. This surgery needs working space for operating the endoscope, but there is no space for it in body cavity. Therefore, we have been developing a surgical tool system which can be inserted from the mouth and ensure space in body cavity. We use magnets and a tool to ensure space, like in abdominal wall lifting method, without use of wire. In this paper, we designed and realized a system which used a tool consisting of link mechanism and magnets. The link mechanism consists of four-bar linkage and a compression spring. It can be driven only by pulling wires, changing the diameter of the tool. We designed this surgical tool prototype, analyzed the stress and did basic experiments.

Levels of thoron and progeny in high background radiation area of southeastern coast of Odisha, India.

Exposure to radon, (222)Rn, is assumed to be the most significant source of natural radiation to human beings in most cases. It is thought that radon and its progeny are major factors that cause cancer. The presence of thoron, (220)Rn, was often neglected because it was considered that the quantity of thoron in the environment is less than that of radon. However, recent studies have shown that a high thoron concentration was found in some regions and the exposure to (220)Rn and its progeny can equal or several time exceed that of (220)Rn and its progeny. The results of thoron and its progeny measurements in the houses of high background radiation area (HBRA) of the southeastern coast of Odisha, India presented here. This area is one of the high background radiation areas in India with a large deposit of monazite sand which is the probable source of thoron. Both active and passive methods were employed for the measurement of thoron and its progeny in cement, brick and mud houses in the study area. Thoron concentration was measured using RAD-7 and Raduet. A CR-39 track detector was employed for the measurement of environmental thoron progeny, both in active and passive modes. Thoron and its progeny concentrations were found to be comparatively high in the area. A comparison between the results obtained with various techniques is presented in this paper.

Distribution of terrestrial gamma radiation dose rate in the eastern coastal area of Odisha, India.

Terrestrial gamma radiation is one of the important radiation exposures on the earth's surface that results from the three primordial radionuclides (226)Ra, (232)Th and (40)K. The elemental concentration of these elements in the earth's crust could result in the anomalous variation of the terrestrial gamma radiation in the environment. The geology of the local area plays an important role in distribution of these radioactive elements. Environmental terrestrial gamma radiation dose rates were measured around the eastern coastal area of Odisha with the objective of establishing baseline data on the background radiation level. The values of the terrestrial gamma radiation dose rate vary significantly at different locations in the study area. The values of the terrestrial gamma dose rate ranged from 77 to 1651 nGy h(-1), with an average of 230 nGy h(-1). During the measurement of the terrestrial gamma dose rate, sand and soil samples were also collected for the assessment of natural radionuclides. The activities of (226)Ra, (232)Th and (40)K from these samples were measured using a gamma-ray spectrometry with a NaI(Tl) detector. Activity concentrations of (226)Ra, (232)Th and (40)K ranged from 15.6 to 69 Bq kg(-1) with an average of 46.7 Bq kg(-1), from 28.9 to 973 Bq kg(-1) with an average of 250 Bq kg(-1) and from 139 to 952 Bq kg(-1) with an average of 429, respectively. The detailed significance of these studies has been discussed from the radiation protection point of view.

Assessment of microsatellite instability status for the prediction of metachronous recurrence after initial endoscopic submucosal dissection for early gastric cancer.

The technique of endoscopic submucosal dissection (ESD) has been developed for en bloc resection of early gastric cancer (EGC); however, little is known about the risk of metachronous cancer in the remnant stomach after initial ESD. In this study, we investigated the correlation between microsatellite instability (MSI) status and the incidence of metachronous recurrence of gastric cancer. According to the genetic/molecular background determined with MSI status and expression levels of hMLH1 and p53 tumour suppressor, 110 EGCs removed with ESD were subclassified into three groups: the mutator/MSI-type (8%), suppressor/p53-type (45%) and unclassified type (47%). Interestingly, patients with the mutator/MSI-type tumour had a high incidence (67%) of metachronous recurrence of gastric cancer within a 3-year observation after initial ESD, which was significantly higher than those with the suppressor/p53-type and unclassified type tumours (P<0.01). Although we investigated mucin phenotypes, there was no correlation between mucin phenotype and the recurrence of EGC. These findings suggest that subclassification of molecular pathological pathways in EGCs is required for the assessment of patients with a high risk of recurrent gastric cancer. The information delivered from our investigation is expected to be of value for decisions about therapy and surveillance after ESD.

Control of intracellular movement of connexins by E-cadherin in murine skin papilloma cells.

The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.

Reduction of malignant phenotype of HEPG2 cell is associated with the expression of connexin 26 but not connexin 32.

Connexin (Cx) genes have a negative growth effect on tumour cells with certain specificity. However, it is not clear whether each Cx gene can act similarly in growth control. Hepatocytes normally express Cx26 and Cx32 as their major gap junction genes, but HepG2 cells, a hepatoma cell line, are deficient in gap junctional intercellular communication (GJIC) based on the down-regulation of Cx26 and aberrant localization of Cx32. In this study, we showed that some of the expressed Cx26 protein in HepG2 cells localized in the plasma membrane and contributed to recovery of GJIC, while the Cx32 protein remained localized in the cytoplasm. The Cx26-transfected clones showed a significantly slower growth in vivo as well as in vitro and reduced anchorage-independent growth ability compared with a mock-transfected clone. Cx26-transfected cells had more regular cell layers due to the re-establishment of the E-cadherin cell adhesion complex. E-cadherin expression following Cx26 transfection was induced. Cx26 expression simultaneously brought E-cadherin and beta-catenin proteins into the plasma membrane without any change in the expression level of beta-catenin protein. These results suggest that the expression of Cx26 contributes to negative growth control of HepG2 cells and the morphological change through the induction of E-cadherin and subsequent formation of cell adhesion complex.

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice.

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.

Connexin43 suppresses MFG-E8 while inducing contact growth inhibition of glioma cells.

Gap junction expression has been reported to control the growth of a variety of transformed cells. We undertook parallel analysis of connexins Cx32 and Cx43 in glioma cells, which revealed potential mechanisms underlying this phenomenon and led to several novel findings. Cx43, but not Cx32, suppressed C6 glioma cell growth. Paradoxically, Cx32 transfection resulted in severalfold more dye transfer than Cx43. However, Cx43 transfectants shared endogenous metabolites more efficiently than Cx32 transfectants. Interestingly, a significant portion of Cx43 permeants were incorporated into macromolecules more readily than those that transferred via Cx32. Cx43 induced contact inhibition of cell growth but in contrast to other reports, did not affect log phase growth rates. Cell death, senescence, or suppression of growth factor signaling was not involved because no significant alterations were seen in cell viability, telomerase, or mitogen-activated protein kinase activity. However, suppression of cell growth by Cx43 entailed the secretion of growth-regulatory factors. Most notably, a major component of conditioned medium that was affected by Cx43 was found to be MFG-E8 (milk fat globule epidermal growth factor 8), which is involved in cell anchorage and integrin signaling. These results indicate that Cx43 regulates cell growth by the modulation of extracellular growth factors including MFG-E8. Furthermore, the ability of a Cx to regulate cell growth may rely on its ability to mediate the intercellular transfer of endogenous metabolites but not artificial dyes.

Comparative PCR: a simple and sensitive method for quantifying low-abundance mRNA species.

To measure low-abundance messenger RNA species comparatively, we developed a simple and highly sensitive quantification method designated comparative PCR. Messenger RNAs from two samples were converted into cDNAs with modified oligo(dT) primers (designated RT primers) containing a sample-specific sequence and a common sequence. After equal amounts of the cDNAs were mixed together, a target gene was amplified by competitive PCR with additional primers: a gene-specific primer and a primer consisting of the common sequence of the RT primers. The amplified products were visualized by the final PCR, designated fluorescence PCR, with an additional three primers: two different fluorescence-labeled primers consisting of the sample-specific sequence within the RT primers and a nested gene-specific primer. Expression levels of the target gene in the two samples were measured by calculating ratios of two different fluorescence intensities. We could quantify 0.1-0.3 copies of the target mRNA per cell from only 0.5 ng of poly(A)(+) RNA for a single detection. This system should be useful for sensitive measurement of scarce transcripts from small samples with a limited amount of RNA such as biopsy specimens.

Induction of skin papillomas, carcinomas, and sarcomas in mice in which the connexin 43 gene is heterologously deleted.

It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of "initiated" cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/- and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7, 12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/- and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/- and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells.

Differences between bulimia nervosa and binge-eating disorder in females with type 1 diabetes: the important role of insulin omission.

This study explored the differences between bulimia nervosa ("BN," n=22) and binge-eating disorder ("BED," n=11) in type 1 diabetic females and the factors most predictive of poor glycemic control in patients suffering from these disorders. These two groups and a control group without eating disorders (n=32) were compared across a number of demographic, psychological, and medical variables. BN manifested significantly more severe disturbances related to eating disorders, depression, anxiety, a higher rate of co-occurring mental disorders, and poorer psychosocial functioning compared with BED. BN also showed poorer glycemic control. Multivariate analysis indicated that higher serum glycosylated hemoglobin (HbA1c) levels were most associated with the presence of severe insulin omission in type 1 diabetic females with binge eating. Clinicians may be able to determine the psychological/medical severity of illness in these patients by identifying the presence of compensatory behaviors to prevent weight gain such as severe insulin omission, as described in the DSM-IV.

Gap junction proteins connexin32 and connexin43 partially acquire growth-suppressive function in HeLa cells by deletion of their C-terminal tails.

Our laboratory has previously reported that transfection of a connexin26 (Cx26) gene, but not connexin40 nor connexin43 (Cx43), into HeLa cells expressing no detectable level of connexins suppressed the tumorigenic phenotype of the HeLa cells both in vitro and in vivo, although all of these connexins induced gap junctional intercellular communication in HeLa cells to a similar extent. The most remarkable structural difference between connexin proteins is the length of the C-terminal cytoplasmic tail, Cx26 having almost no tail, while Cx43 and connexin32 (Cx32) have long and intermediate ones, respectively. When Cx32 and Cx43 lose their C-terminal tails, they seem to resemble Cx26 in structure. To examine whether such truncated connexins become tumor suppressive in HeLa cells, we introduced a stop codon into each of the Cx32 and Cx43 cDNAs to remove their C-terminal tails and transfected these constructs (DeltaCx) into HeLa cells. Both DeltaCx cDNAs induced GJIC as efficiently as the wild-type counterparts. Although none of the truncated connexins affected proliferation rate, the truncated Cx32 and Cx43 proteins suppressed anchorage-independent cell growth in soft agar. Furthermore, when the transfectants were injected into the backs of nude mice, tumor appearance was delayed by 7 days in animals given cells expressing truncated connexins, i.e. tumors became detectable on days 11 and 18 after injection of vector and DeltaCx transfectants, respectively. Although throughout these experiments the truncated connexins did not completely eliminate the tumorigenicity of HeLa cells, as Cx26 did, it was evident that deletion of the C-terminal tails gave both Cx32 and Cx43 a capacity for negative growth control, suggesting that the C-terminal tails of these two connexins function as a regulatory region for connexin-mediated growth control in HeLa cells.

Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis.

During multistage carcinogenesis, the functions of several key genes involved in cell growth control must be damaged. Such genes include not only those involved in cell cycle control of individual cells, but also those involved in the coordination of cell growth throughout a given tissue through cell-cell communication. The most intimate form of intercellular communication is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is known to transfer small water soluble molecules, including cAMP and IP3, from the cytoplasm of one cell to that of its neighbors; the growth of a given GJIC-associated cell is thus kept in check by other GJIC-connected cells. Most tumor cells have a reduced ability to communicate among themselves and/or with surrounding normal cells, confirming the importance of intact GJIC in growth control. When connexin (gap junction protein) genes are transfected into such cells, normal cell growth control is often recovered. Certain dominant-negative mutant connexin genes can reverse such tumor suppression. While these results suggest that connexin genes form a family of tumor suppressor genes, so far we have found no connexin gene mutations in human tumors; only two connexin gene mutations were found in chemically induced rat tumors. On the other hand, our recent studies suggest that connexin genes may be inactivated by hypermethylation of their promoter regions, suggesting that epigenetic inactivation of connexin genes may be a mechanism of GJIC disturbance in certain tumors. However, in many tumor cells connexins are normally expressed but aberrantly localized. The mechanisms of aberrant localization of connexins include lack of an appropriate cell-cell recognition apparatus and aberrant phosphorylation of connexins. These results suggest that GJIC disorders may occur not only because of aberrant expression of connexin genes themselves, but also as a result of disruption of various control mechanisms of the protein functions.