富山医科薬科大学医学会

Other富山医科薬科大学医学会誌, 3(1), 1990.03, pp.56-61othe

University of Toyama Repository

富 山 医薬大医誌 3 巻 1 号 1990年第 4 回 富 山 医 科薬科大学電子顕微鏡懇話会 （抄録）日 時 ： 平成 2 年 3 月 2 日 幽会場 ： 附属病 院臨床第 二講義室特別講演Cel l ular sites of action of steroid hormones Use of autoradiography for their identification Walter E. STUMPF Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC, U. S. A Autoradiographic studies with radiolabeled steroid hormones have provided informat10n on sites of action, which were difficult to identify by disruptive biochemical approaches. Due to the high sensitivity of the autoradiograph1c method and the possibility to study relatively mtact tissues, information on individual target cells within the topographical context has been obtained. Thus, such data have expanded our knowledge base for estradiol, progesterone, testosterone, and adrenal steroids. In recent years, extensive new information became availa­ble on vitamin D metabolites, demonstrating nuclear receptor sites for 1, 25-dihydroxychole­calciferol in many tissues that include B-cells of the endocrine pancreas to promote insulin secretion ; TSH-cells in the anterior pituitary to promote TSH secretion ; gastrinーproducingcells in the antrum of the stomach , cells of the adrenal medulla, both epinephrine and nor­epinephrine producing cell types ; atrial card10 myocytes, colocal ized with atrial natriuretic factor (ANF), probably to influence the secre­tion of ANF ; cells in the epidermis and hair sheaths to promote cell proliferation and dif­ferentiation (as subsequently shown by Dr. Suba at Showa University) ; Sertoli cells of the testis ; certain cells in the epididymis and prostate ; germinal epithelium of the ovary ; certain cells in the oviduct , ductal and acinar epithelium in the mammary gland ; chief cells in the para­thyroid ; reticular cells in the thymus and spleen, as well as certain macrophages in lymph nodes and spleen ; myoepithelial cells in salivary glands ; and neurons in brain, spinal cord, and spinal ganglia. While some of the functions of the new target tissues have been clarified, much work will be necessary in order to learn about the functions of vitamin D at the newly identified sites of genomic action. Of a particular challenge are the many sites described in the brain. In the brain, vitamin D receptor neurons are seen especially in the central nucleus of the amyg­dala and in the bed nucleus of the stria termi­nalis, in the reticular nucleus of the thalamus, m the periventricular nucleus of the hypo­thalamus, in the piriform cortex, in area CA4 of the hippocampus, in the raphe nucleus of the m1dbrain, and in motor nuclei of cranial nerves The topographic distribution of vitamin D target cells in the central nervous system sug­gests that vitamin D is involved in the regula­tion of all neural functions that include autono・m1c, neuroendocrine, sensory, and motor act1v1-ties. From the new information derived from the autoradiographic studies and the present know­ledge about related functions, it can be stated that vitamin D is a steroid hormone (not a v1-tamin !) that has extensive influences on select target cell populations throughout the body. In po phυ its actions and distribution, vitamin D resembles estradiol and other steroid hormones. Vitamin D, which we call more appropriately SOL TRIOL (Stumpf, 1988), is the steroid hormone of sunlight. With the seasonal rise and fall of soltriol blood levels, the activities of all vital systems in the body are regulated and adjusted to the seasonal environment in order to assure maintenance of life, with optimal seasonal adaptation of growth and reproduction. Accordingly, vitamin D-soltriol is involved not only in the regulation of calcium levels, as was believed for a long time, but also m the regulation of reproduction, thyroid func­tion, carbohydrate metabolism, immune response, mental processes, cardiovascular function, and the entrainment of biorhythms. 一般講演I . App l ication of rad ioisotope-labeled l ipid microspheres as a drug del ivery system­where are microspheres found in  EM ? Takashi HONDA Radioisotope Laboratory, Toyama Medical and Pharma­ceutical University Artificial lipid microspheres (LM-0.2µm in aver­age diameter) containing 14C-palmitic acid label­ed triglycerides were injected intravenously into rats of the carrageenan paw edema tests. LM m the inflamed paw skin was observed to be ap­proximately 2 to 3 times higher localized than that in the non-inflamed skin. With the electron microscope, LM were found to be diffused into exudate in inflamed rat paw. Electron micrographs of the inflamed tissue showed that the endothelial cells of blood vessels contained LM and that LM penetrated from basement membrane to outer layer of the vessels and localized in the inflamed tissues. Many LM were taken up by macrophages. - 57 Therefore, the results of the electron microscopy study support the pharmacokinetic findings on tissue distribution of LM, and provide additional evidence for uptake in endothelial cells, and ma­crophages and for penetration into extravascular exudate. 2. The e lemental composition of cel l  organel les as studied by X-ray microanalysis using fresh frozen dried ultrathin sections Kenichi TAKAYA and Tutomu ISHIKURO Department of Anatomy, Toyama Medical and Pharma­ceutical University Myelin of several vertebrates was studied by a quantitative X-ray microanalysis using fresh frozen dried ultrathin sections. The mesence­phalon of a dog and a cat, the cerebral cortex of rats and the spinal cord of tree frog tadpoles were freshly frozen in freon precooled with liquid nitrogen. Ultrathin sections (lOOnm) were attached on the titanium grids treated with collodion and freeze-dried in nitrogen gas flow. The sections were firstly observed by 200 kV electron microscopy and then microanalyzed with the analytical electron microscope (X-650) on the STEM mode at an acceleration voltage of 40 kV , a specimen current of 2nA and count­ing time of lOOs. A quantitation was made by the P/B ratio method and the concentration was expressed as mM/kg dry weight. The standard solutions were prepared by dissolving compounds of 16 elements including sodium, magnesium, aluminum, silicon, phosphorus, sulfur, chloride, potassium, calcium, manganese, iron, chromium, nickel, copper, zinc and barium in 20% polyvinyl­pyrrolydone (PVP-K30). The myelin sheath and the surrounded axon were well preserved and identified clearly. However, laminated figures of the myelin sheath were not discernible even at a high magnification. High amounts of phos­phorus and potassium were disclosed in myelin but they showed a great variation among the regions of the brain. Phosphorus, 408 mM/kg dry weight and potassium, 408 mM/kg were disclosed in the dog myelin and phosphorus, 380 and potassium, 256 in the cat myelin, respective­ly. A higher concentration of phosphorus was detected in nuclear regions than in myelin. 3. Transformation of transplantable Mastomys’ gastric carcinoid cel ls into rhabdomyocytes Y oshiaki KARAKI and Masaru SAWATAISHI Second Department of  Surgery, Toyama Medical and Pharmaceutical University We have successfully maintained transplantable Mastomys’ carcinoid in the subcutaneous reg10n of the thigh of recipient mastomys ; the orginal tumor was spontaneously produced in the Masto­mys’ glandular stomach of the Niigata strain. To cl arify the relationship between stromal tissue especially, capsular structure and component cells of this particular neoplasm, histologic, his­tochemical and electron-microscopic studies were undertaken. Tissues were taken from the transplants of the 15th generation of the Niigata strain. Histologically and histochemically, tumors were usually surrounded by loose con­nective tissue or so-called pseudocapsule contam­ing some kinds of stromal cel ls. These capsular stromal tissues often have a continuity to the mtraneoplastic stromal tissue dividing it into several neoplastic islands or nests. In these capsular stromal tissues and nearby stroma adjacent to the peripheral neoplastic nests of the main tumor mass, typical rhabdomyocytes were frequently seen. Electron-microscopically the capsular structure was in some part composed of elongate rhabdomyocytes. In some neoplastic n6 whd nests nearby the capsular structure and stroma around the nests, immature, transitional or ma­ture type of rhabdomyocytes were frequently observed. Furthermore, some of the immature cells possessed both myofibrils with z band and endocrine granules. These findings strongly sug­gest that Mastomys’ gastric carcinoid cells in transplants have a possibility of being trans­formed into rhabdomyocytes resulting in makmg a capsule of the tumor. 4. Ultrastructures of experimental thymoma Shigeki SUGIYAMA, Toru TAKANO and Yang WANG First Department of Surgery Toyama Medical and Phar­maceutical University The fine structures of human and experimental thymoma and various carcinomas have been documented. We have observed a spontaneous thymoma in Buffalo rats, 12 months old. An attempt was made to obtain the thymoma m a short exposure to N-nitroso-N-methylurea(NMU) combined with N-bis (2-hydroxypropyl) nitro­samine (BHT). The electron microscopic study of the induced tumors was made. Twenty four newborn mice were divided into two groups. Group 1 : A single high dose of NMU (600mg/kg) was administered subcutane­ously in ten mice. Group 2 : A single high dose of NMU combined with total seven injections of BHT (250mg/kg per time) in 1 4  mice. The am­mals were maintained at room temperature(22℃ ） and given food and water ad libitum. Two mice of group 1 and six mice of group 2 survived at 18 weeks after the administration of the carcino­gens. The necropsy was performed to all the mice that died or were killed at the regular inter­vals. The thymomas and other tumors were fixed m 4 %  paraformaldehyde solution and stained with Hematoxylin-Eosin. Other small blocks of tumors were fixed with 2% glutaraldehyde in phosphate buffer, postfixed in 3% osmium te­troxide in phosphate buffer, and embedded m epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under H-300 electron microscope. Tumors developed in 10 to 15 mice (66 .6%) in 18  weeks after the single NMU injection. No tumor was found in group 1 mice surviving for 18 weeks, but four of  the six surviving mice of group 2 had tumors. All thymomas were identified as a lymphoid cell type microscopically. In the tissues, scarce epithelial reticular cells containing well -devel­oped ton of ilaments were seen but a large number of lymphocytes were observed 5. Ultrastructural study on secretory glyco・conjugates of pancreatic cancer cel ls Nobuyuki NISHIMURA, Yoshiki KUBOTA, Seizi SAITO, Kyoichi INOUE and Akihara WATANABE Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University The pancreatic carcinoma develops from the duct epithelium of the pancreas which has an ability to produce secretory conjugated sugars. Using the pancreatic cancer cell line derived from well differentiated adenocarcinoma, binding of lectins by the glycoconjugates of the cells was studied by light and electron microscopy. Cancer cells were isolated from the ascites of the car­cinomatous peritonitis patient and the cell !me of HuP T4 was established by subculture. Tumor tissue specimens obtained by inoculation of HuP­T 4 cells into the subcutis of nude mice were used for this study. Seven lectins were used ； 羽「GA,RCA-1 ,  PNA, Con A, UEA-1,  SBA and DBA. They were applied by direct and indirect me­thods. By light microscopy. WGA, RCA-1 ,  PNA and UEA-1 stained the luminal cell border and the mucoid substance intensely and the cyto­plasm in the intermediate degree. Con A was bound chiefly in the cytoplasm and no staining with DBA was shown. By electron microscopy, WGA, RCA-1,  PNA and UEA-1  were localized at microvilli, secretory granules and Golgi ap­paratus. At the lateral plasma membrane, the reactions with WGA, RCA-1 and UEA-1 were revealed but no reactions with PNA. SBA show­ed an intense reaction at microvilli and secreto­ry granules but DBA had no reaction. From the above findings, the secretory glycocon­JUgates produced by HuP-T4 cell line have N­acetylgalactosamine (or sialic acid). D-galactose and fucose. Discrepancies in the distribution of the reactions with SBA and DBA both of which have a specificity to N-acetylgalactosamme are interesting. 6. The reaction of sera from patients with m ixed connective tissue disease with acid phosphatase positive phagocytes in the red pulp of the mu­rine spleen Tetsu KAMITANI*, Saburou YANO*, and Kenichi TAKAYA* First Department of Internal Medicine*, Second Department of Anatomy, Faculty of Medicine** Toyama Medical and Pharmaceutical University In the immunohistochemical study, we found that sera from patients with certain rheumatic diseases, particularly MCTD (mixed connective tissue disease), reacted with acid phosphatase positive phagocytes in the red pulp of the adult murine spleen. The reaction was due to Fab fragments of the IgG antibodies in the patients sera. The reactive antigen proved to be N aI04 nu 伊hdand RN ase sensitive, suggesting it might be a RNA-associated antigen. What is this antigen ? As a working hypothesis it was assumed that it might be a viral antigen, because it was RN ase sensitive, and because almost all patient sera with the antibodies also contained the anti-U1 -RNP antibody which reacts with a retroviral gene product. In the present study, we examined the red pulp macrophages of the murine spleen by using electron microscopy. In the cytoplasm of the macrophages, virus-like particles were ob­served. It is not known at present, however, whether or not these particles were related to the antigen reacting with the patient sera. Im­munoeleetron microscopic studies will be neces­sary to ascertain the relat10n. 7. lmmunocytochemical study of S・ I 00 protein positive adherent cel ls obtained from human tonsils Makoto KAWAGUCHI*, Shin ISHIZAWAへFuyumi SHIMO DA＊ぺ Hiroshi ODAKE**, Takeshi SAKAI* and Fumitomo KOIZUMI* Department of  Pathologyへ Department of  Oral and Maxillo Facial Surgery**, Section of Japanese Oriental Medicine***, Toyama Medical and Pharmaceutical Uni­versity Hospital Both macrophages (M ¢) and dendritic cells (DC) are of a plastic adherent nature. We examined human tonsillar transiently adherent cells (TAC) using immunocytochemical double staining tech­nique. Immunoreactivity for monoclonal anti­bodies [Anti-LeuM5 (CDl lc), DAKO・Macrophage,FMC32, Myeloid/Histiocyte Antigen (DAKO­MAC387) ] was investigated on S-100+ TAC. ．S-lOO+ TAC were on the average 97% CDl lc� 86% DAI王ODAKO-MAC387+. The distribution patterns of the above mentioned antibodies-positive cells m -60 the tonsil tissue were also examined. In the T­cell-dependent area, S-100+ interdigitating cells (IDC) and MAC387+ M¢ were present. In addi‘ tion, many scattered CDl lc+, DAKO-Macro­phage+, FMC32+ cells were observed, each m a separate staining. ＼九Tith regard to the staining overlap in the analysis of TAC and distribution patterns of M¢ and DC in the tonsil tissue, we reached two conclusions. First, most of the S-100+ IDC in the T-cell-dependent area are im­munoreactive for CDl lc, DAKO Macrophage and FMC32. Second, DAKO・MAC38r M¢ and S-100+ DC are completely separate cellular popu­lations 8. Findi ngs in pericardia!  pore formarmation Y oshifumi FUKUO Department of Anatomy Toyama Medical and Pharma­ceutical University I have described the pericardial pores which commumcate the pericardial cavity with pleural cavities. In order to investigate the formative process of the pores, the pericardium of the ddY mice from birth to 21 days was examined. Up to 6 - 7  postnatal days, the mice pericardium consisted of the mesothelial cell layer of the parietal pericardium, the mesothelial cell layer of the parietal pleura and the connective tissue sandwiched between them. Abundant pericardial pores made their appearance at 1 0 - 20 days after birth, cell processes from the mesothelial cells of one side towards those of another side developed to form cellular junctions connecting from the side developed to form cellular junctions connect heart side to the lung side. As a result, pen­cardium made tongue- like processes connected with each other at the tips. When the junctional contact of the processes separated, the pencar­dial pores were produced 9. Scanning electron microscopic study of p i l i  torti (twisted hairs) Tomohiro MARUYAMA, Akihiro KANEI, Shozo TAKAHASHI and Masaaki MOROHASHI Department of Dermatology Toyama Medical and Phar maceutical University We observed a case of the twisted hair of a sixteen year old I apanese girl who had suffered from large area of scanty, short and frizzled hairs by birth. We examined the specimen taken from her occipital region by light and scanning electron microscopy and obtained the following findings ; 1) A part of the hair shaft is flattened and the hair is twisted clockwise and anti-clockwise on its own axis. 2) Some hairs are knotted. 3) A knot develops at the twisted region. 4) Hair cuticles are easily dropped out by a friction. 5) Hairs were found which have longitudinal ruptures along the hair axis. 6) Morphological changes of the hair begin at the hair root. 7) Short hairs have a disruption at the flattened region, at the unflattened region, and at the knot and at the root of ”branched hairs”． 8) Light microscopy revealed a vacuolation of the cells and an irregularity of thickness in outer root sheath at the hair bulb. 9) Their upper part showed a morphological change at outer root sheath, inner root sheath and the lumen of the hair follicle. 1 0) There is a morphological change of the duct of the sebaceous gland. 1 1) By DAC恥f staining, although there was no change in the hair shaft and inner root sheath, some eosinophilic pyknotic cells in the outer root sheath revealed abnormal conversion of SH groups to SS linkages in cellular protein. From the above findings, the mechanism of the development of the morphological change of the twisted hairs of the present patient is caused by 1) Unequal development of the cells at the outer root sheath at the hair bulbs inducing a marked change of the inner root sheath and hair shaft in the upper part leads to the twisted hair. 2) Friability of the hair is chiefly due to its morphological changes. 唱EAnhu 

第4回富山医科薬科大学電子顕微鏡懇話会（抄録）

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第4回富山医科薬科大学電子顕微鏡懇話会（抄録）

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