Search

The purpose of this study was to compare the biological effects of fractionated doses versus a single dose of high-LET carbon ions in bystander normal cells, and determine the effect on their progeny using the layered tissue co-culture system. Briefly, confluent human glioblastoma (T98G) cells received a single dose of 6 Gy or three daily doses of 2 Gy carbon ions, which were then seeded on top of an insert with bystander normal skin fibroblasts (NB1RGB) growing underneath. Cells were co-cultured for 6 h or allowed to grow for 20 population doublings, then harvested and assayed for different end points. A single dose of carbon ions resulted in less damage in bystander normal NB1RGB cells than the fractionated doses. In contrast, the progeny of bystander NB1RGB cells co-cultured with T98G cells exposed to fractionated doses showed less damage than progeny from bystander cells co-cultured with single dose glioblastoma cells. Furthermore, inhibition of gap junction communication demonstrated its involvement in the stressful effects in bystander cells and their progeny. These results indicate that dose fractionation reduced the late effect of carbon-ion exposure in the progeny of bystander cells compared to the effect in the initial bystander cells.

血管内皮細胞による血管運動調節では、内皮細胞から放出された液性物質がpharmacomechanical couplingによって血管平滑筋の収縮に影響する機構がまず発見されたが、内皮細胞の膜電位変化により平滑筋の膜電位が変化し、それがelectromechanical couplingにより収縮・弛緩を引き起こす機構も存在する。アゴニスト刺激によって内皮細胞内Ca2+濃度が増加するとCa2+活性化K+チャネルが素早く、Ca2+活性化非選択性陽イオンチャネルが徐々に開き、前者によりまず膜の過分極が、後者により遅発性の膜の脱分極が生じる。この内皮細胞の膜電位変化が平滑筋一内皮細胞間ギャップ結合を通って平滑筋に伝わる。平滑筋の電位依存性Ca2+チャネルは過分極によって閉じ、脱分極によって開くので、これによって平滑筋の細胞内Ca2+濃度が変化し、収縮が制御される。この膜電位による血管運動調節は薄い平滑筋層を持つ細動脈で特に有効で、その部で決定される末梢血管抵抗の調節に重要と考えられる。, Vascular endothelial cells control the vasomotor responses in two mechanisms. An agonist stimulation applied to the endothelium increases the endothelial intracellular concentration of Ca2+, which triggers a release of vasoactive substances such as NO and PGI2. These substances diffuse to the smooth muscle cells and modulate their mechanical activities by the pharmacomechanical coupling. Increased endothelial intracellular Ca2+ also activates the Ca2+-activated K+ channels quickly and the Ca2+-activated non-selective cation channels gradually evoking endothelial hyperpolarization and depolarization, respectively. These changes in endothelial membrane potential conduct to the smooth muscle cells via the myoendothelial gap junctions and the contraction of the smooth muscle cells is modulated by the electromechanical coupling. As the electromechanical mechanism is most effective in arterioles which have only one or two layers of smooth muscle cells, this mechanism seems to be crucial in controlling the total peripheral resistance that is mainly determined by this portion of vasculature.

Tight junctions are intercellular junctions adjacent to the lateral end of the apical membrane. They have two functions: the barrier （or gate） function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea and blood-borne metastasis. The blood-brain barrier exclusively depends on this function of tight junctions of endothelial cells. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membranes. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. We have been trying to expand our understanding of molecular regulation of tight junctions to human diseases. In this review, we introduce three projects currently ongoing; studies on tight junctions of hepatocytes deeply related to jaundice, tight junctions of endothelial cells crucial for the blood-brain barrier, and the formation and regulation of tight junctions during establishment of cell polarity.

We have begun to investigate cellular bystander responses in normal human fibroblasts induced by microbeams of different radiation types. This year, we used two different microbeams, such as carbon ions as high-LET (Linear Energy Transfer) radiations and X rays as low-LET radiations. Carbon-ion microbeams ((12)C(5+), 220 MeV) were generated with the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) at Japan Atomic Energy Agency. Mono-energetic X-ray (5.35 keV) microbeams were produced with the Photon Factory (PF) at High Energy Accelerator Research Organization. We designed that only 0.2 percent of total cells was irradiated with each microbeam. The percent cell survival in microbeam-irradiated dishes was ranging from 83 percent to 94 percent for carbon ions, while around 100 percent for X rays. Mutation frequency for carbon-ion-microbeam irradiated cells was about 6 times higher than that for non-irradiated control cells, but there was no difference in mutation frequency between X-ray-microbeam irradiated and control cells. There is clear evidence that bystander cellular effects induced by low-dose radiations depends on a radiation quality., 資料番号: AA0063706092

Mammalian brains consist of a variety of cells such as neurons,astrocytes,oligodendrocytes and vascular endothelia. It is believed that appropriate cell- cell interaction is essential for the development of the mammalian brain. To clarify changes in gap junctional intercel-lular communication during the early stages of neural differentiation,we established an in vitro system for neural induction from mouse embryonic stem （ES） cells and analyzed their capability of gap junctional intercellular communication by microinjection tracer coupling with neurobiotin.Undifferentiated ES cells were culturedirL SuSpenSion for8days and treated with all-trans retinoic acid（500 nM） during the latter half of tlle period. Thereafter,Cells were transferred to adhesion culture. Neurites were observed in more than 90% of embryoid bodies at day 6 after the beginning of adhesion culture. Concerning neural markers, in small clusters at day 1 of adhesion culture cells were positive for A2B5, which is known to be expressed from the neuroepithelial stage in vivo. Cells with neurites showed positive signals of microtuble-associated protein 2（MAP2）and neurofilament M subunit, markers for more differentiated neurons on and after day 3 of adhesion culture. Utilizing this in vitro neural-differentiation system from ES cells, We dispersed embryoid bodies by treatment with trypsin and developed a dispersed cell culture system for early neuronal cells. In this system,cells positive for neuronal markers formed small colonies after one day of culture. Neurobiotin microinjected into a single cell was found to spread into all cells constituting colonies that expressed A2B5 at day 1 of adhesion culture after dispersion, but the dye did not spread into flat cells when there was no A2B5 expression around the colony. On the other hand, after day 3 of adhesion culture after dispersion, neurobiotin injected into a single cell in the colony was restricted to small portions of the colony. Connlexin26 was expressed in neural cells within the colony. These results suggest that compartmentalization of gap junctional communication was stabiished between neuron cells and other cell lineages and that the capability of communication within neural cells decreases rapidly during the process of neural differentiation.

We have reported the efficacy of intra-arterial-combined immunochemotherapy including interleukin2 (IL-2) for unresectable hepatocellular carcinoma (HCC). To further test this therapy for prevention of intrahepatic recurrence after hepatectomy, the influence of IL-2 on liver regeneration was examined using the bromodeoxyuridine (BrdU) labeling index (LI) in 70% hepatectomized Donryu rats. In addition, gap junction appearance was analyzed using a monoclonal antibody (HAM 8). IL-2 (45,000 JRU/day) or saline was administered continuously via the portal vein immediately after hepatectomy. We also examined the influence of IL-2 on liver regeneration after hepatectomy with splenectomy. No difference in the weight of the liver, Serum albumin, GPT, or total bilirubin was observed in any groups at 1, 2, or 4 days after hepatectomy. Neither IL-2 nor splenectomy influenced BrdU LI at all three points. Gap junctions began to disappear after hepatectomy and reached a minimum on day 2 in all groups. Four days after hepatectomy, the density of the reappearing gap junctions was markedly lower in groups treated with IL-2 than in those receiving saline with or without splenectomy. However, the density at 6 days after hepatectomy in all groups was identical to the density in normal liver. Continuous portal infusion of IL-2 transiently disturbed gap junction reappearance during liver regeneration. However, no other parameters of liver regeneration or liver functions differed. These results suggest that intrahepatic IL-2 administration can be performed without serious complications after hepatectomy.

Gap junctions are channels through which ions and small molecules are directly exchanged between adjacent cells, and consist of connexon. Connexon is composed of 6 connexin （CX） molecules, of which CX32 and CX26 have been isolated from the rat liver. To study distributional changes of CX32 and CX26 in rat hepatocytes in the developmental, regenerating and carcinogenic processes, we performed immunohistochemical analysis using a new polyclonal antibody, anti-CX26 antibody, and an antibody which we reported on previously, anti-CX32 antibody, both raised against synthetic peptides of the intracytoplasmic domain. With liver maturation, the CX32 positive-spots （CX32-PS） remained evenly distributed throughout the acinus of the liver and increased in number. However the CX26-PS, which were sparsely distributed in the embryonic stage, became localized specifically in the periportal area without an increase in number. Thus, we suppose that the alteration of localization of CX26 might be associated with maturation of the liver. In rat livers regenerating after partial hepatectomy, changes in the number of CX26-PS were similar to those of CX32-PS. This result suggests that these CXs might be controlled by the same mechanism. In preneoplastic lesions induced by the method of Solt and Farber, the number of CX32-PS decreased, whereas the number of CX26-PS increased in about half of the lesions. In hepatocellular carcinomas, the numbers of both CX26- and CX32-PS decreased. These results suggest that the expression of CX32 and CX26 might be regulated differently during hepatocarcinogenesis. CX32 and CX26 mostly showed similar patterns in the immunofluorescent study. In preneoplastic lesions, however, the two CXs showed obvious differences in their numbers. The significance of the quantitative changes of gap junctions during hepatocarcinogenesis has yet to be investigated.

Gap junctions are specialized membrane channels comprised of 6 connexin molecules and play an important role in intercellular communication between cells in contact. Connexin 32（Cx 32） has been isolated as the major hepatocyte gap junction protein. To study the changes of gap junctions in hepatocytes under various pathologic conditions, we developed a polyclonal antibody against synthetic peptides of the intracytoplasmic domain of the Cx 32 molecule. The specificity of the antibody was confirmed by immunoelectronmicroscopy and Western blot analysis. In order to examine the relation between Cx32 expression and hepatocyte proliferation, immunofluorescent examination of Cx32 in nomal and regenerating liver of rat and mouse was performed. Normal rat and mouse hepatocytes showed around 10 dot-like positive stainings on the cell membrane, however, staining of Cx 32 gradually decreased and almost disappeared in accordance with stage of DNA syn- thesis in regenerating liver of both rat and mouse. Northern blot analysis revealed that a decrease of Cx32 mRNA had already occurred before DNA synthesis. Sequential changes in the staining of Cx 32 in cultured hepatocytes after plating were also examined. The Cx32 positive dots gradually decreased in number and disappeared at 24hr after plating. We examined the effects of the antibody on intercellular communication by microinjection of the antibody and Lucifer Yellow in couplet he- patocytes. The results demonstrated that the injection of antibody inhibited intercellular communi- cation between couplet hepatocytes, suggesting the importance of the intracytoplasmic domain of Cx 32 for regulation of gap junction function.

Cells communicate with each other by gap junctions which are composed of channels that directly connect the interior of interacting cells. Such junctions are widely distributed in tissues of all animals and couple the cells both electrically and metabolically by allowing small molecules to pass from the cytoplasm of one cell into the cytoplasm of another. The presence of gap junctions can be demonstrated morphologically by electron microscopy and functionally by dye transfer, metabolic coupling and electric coupling methods. Howerer, the observation of gap junctions in tissues has been limited to electron microscopy, because the above functional methods are applicable only to the cultured cells. In this study, we developed an immunohistochemical method for visualization of gap junctions in tissues. Gap junctions were successfully isolated from rat liver plasma membranes by a combination of NaOH treatment, sonication and sucrose gradient centrifugation. The isolated gap junctions were morphologically intact, and they were comprised of a 26,000 protein on SDS-PAGE. Antibodies to gap junctional protein were raised in mice, and immunostaining was performed using the antibodies on various tissues including normal, preneoplastic and neoplastic tissues of rodents and humans. The antibodies reacted to the livers of all the species examined, and to rat kidney, stomach, pancreas and salivary glands. It was found that there was a significant difference in the number and size of gap junctions among the tissues. It was noteworthy that the number of gap junctions were markedly decreased in chemically-induced hyperplastic nodules and hepatocellular carcinomas in rats, indicating that cellular communication is reduced during carcinogenesis. The results of this study demonstrated that this immunohistochemical method is useful in quantitating gap junctions in tissues of various kinds of animals including humans.