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Transplantation Proceedings 03/2003; 35(1):573-4. · 1.00 Impact Factor
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ABSTRACT: Angiogenesis is an important event in tumor growth. We evaluated the contribution of endogenous bradykinin to tumor-associated angiogenesis and tumor growth using pharmacological approaches in mice bearing sarcoma 180 cells. The weight of implanted tumors increased in parallel with increased hemoglobin contents (a parameter to evaluate angiogenesis) over a 20-day experimental period. Daily administration of bradykinin B2-receptor antagonists, Hoe140 (0.1 and 1 mg/kg per day, local injection) or FR173657 (30 mg/kg per day, p.o.), significantly suppressed the increment in angiogenesis and tumor weight, but a B1-receptor antagonist, desArg10-Hoe140 (1 mg/kgperday), did not. Administration of a plasma kallikrein inhibitor, soybean trypsin inhibitor (3 mg/site per day), significantly suppressed angiogenesis and tumor growth. In contrast, bradykinin-degrading enzyme inhibitors, captopril and phosphoramidon (500 microg/site per day), enhanced angiogenesis and increased tumor weight. Our results suggest that bradykinin, produced by plasma kallikrein or plasma kallikrein-like enzymes, promote tumor-associated angiogenesis and tumor growth in vivo.
The Japanese Journal of Pharmacology 01/2002; 87(4):318-26.
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ABSTRACT: The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.
Cell Structure and Function 01/2002; 26(6):685-91. · 2.29 Impact Factor
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ABSTRACT: The 3-dimensional shape of the human saccular macula and its orientation in the skull were quantitated in this study. The semicircular canals and saccular maculae were reconstructed 3-dimensionally on a computer from 3 human temporal bones. The 380 to 522 triangles in the entire area of the saccular macula were made by drawing lines between 2 adjacent points every 100-pm width of the saccular macula in each section. The angles between each triangle and each estimated standard axis in the skull obtained were calculated. This information will provide standard data regarding the 3-dimensional morphological aspects of the saccular macula for further investigations of the function of the sacculus. It was determined that the 3-dimensional shape of the saccular macula was not a plane, but was a curved surface like that of an ellipsoid. It is thought that this shape is necessary in order for the saccular macula to detect wide-range linear acceleration.
The Annals of otology, rhinology, and laryngology 12/2001; 110(11):1017-24. · 1.05 Impact Factor
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ABSTRACT: We previously reported that endogenous prostaglandins (PGs) may increase cAMP facilitated angiogenesis through the induction of vascular endothelial growth factor (VEGF) in rat sponge implantation models. In the present experiment, we tested whether or not adenylate cyclase / protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by 8-bromo-cAMP, forskolin and amrinone. Angiogenesis without topical injections of the above compounds was suppressed by SQ22,536, an inhibitor for AC, or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. PGE2-induced angiogenesis was suppressed with SQ22,536 or H-89. These results suggested that AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.
The Japanese Journal of Pharmacology 12/2001; 87(3):181-8.
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ABSTRACT: Because little has been known about the morphological and functional consequences of liver transplantation on hepatic autonomic nerves, we examined the time-course of extrinsic hepatic innervation at the level of the porta hepatis of liver allografts.
Orthotopic liver transplantation was performed using male Lewis rats. Crosscut tissue specimens were obtained postoperatively for up to 6 months from the porta hepatis of transplanted livers, and processed for immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP-43), and for transmission electron microscopy (TEM).
Extrinsic nerve fibers at the porta hepatis stained positively for PGP 9.5 throughout the entire study period. In contrast, the immunoreactivity of GAP-43 was negative at postoperative day (POD) 1 and 2. GAP-43-positive nerves were first observed to appear in the porta hepatis at POD 3. The immunoreactivity of GAP-43 remained positive thereafter until 3 months post-OLT, and became negative in all the specimens at 4 months post-OLT. Transmission electron microscopy demonstrated a small number of regenerating axons existing among many degenerating axons at POD 3. At 3 months post-OLT, most regenerating axons had been fully ensheathed by the cytoplasm of Schwann cells, although their density remained at a lower level compared with normal.
The results of this study suggest that liver allografts become extrinsically reinnervated, with the regenerating axons reaching the hepatic hilus 3 days after transplantation. The process of extrinsic hepatic reinnervation is considered to almost terminate 4 months after transplantation in rats.
Liver International 11/2001; 21(5):300-8.
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Transplantation Proceedings 12/2000; 32(7):1634-6. · 1.00 Impact Factor
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ABSTRACT: We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.
European Journal of Morphology 11/2000; 38(4):237-41.
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ABSTRACT: The ultrastructure of mucous cells of rat sublingual gland processed by rapid freezing, followed by freeze substitution, was compared with that obtained by the standard chemical fixation technique. The rapid freezing method gave a very good preservation of membrane structure with round and discrete mucous droplets (granules) not showing any sign of coalescence. The cisterns of the Golgi apparatus and the trans Golgi network also were well preserved. Upon secretory stimulation by pilocarpine, mucous droplets were discharged by the usual mechanism of exocytosis. From all these findings it emerged that mucous cells had the same structural characteristics as serous cells. In the endpieces of rat sublingual gland prepared by the rapid freezing method, serous cells aligned with mucous cells around the central lumen, and no cap-like arrangement of serous cells (demilunes) was observed. Furthermore, computer reconstruction of stereo images from serial section light micrographs prepared by the rapid freezing method showed that, within a given endpiece, all serous cells had direct access to the lumen and that they were disseminated throughout it and not only in its fundus. From our observations it seems very likely that, at least in rat sublingual gland, serous demilunes are an artificial product caused by the compression exerted on serous cells by the mucous cells distended during the conventional fixation procedure.
European Journal of Morphology 11/2000; 38(4):213-8.
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ABSTRACT: Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo-oxygenase (COX)-2 mediated angiogenesis in chronic and proliferate granuloma. In rat sponge implants, angiogenesis was gradually developed over a 14-day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF). In sponge granuloma, mRNA of COX-1 was constitutively expressed, whereas that of COX-2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF). Topical injections of bFGF increased the expression of COX-2 mRNA. bFGF-stimulated angiogenesis was inhibited by indomethacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. The levels of PGE(2) and 6-keto-PGF(1alpha) in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS-398. The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS-398. Topical injections of PGE(2) and beraprost sodium, a PGI(2) analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement. The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti-sense oligonucleotide. These results suggested that COX-2 may enhance bFGF-induced neovascularization in sponge granuloma by PG-mediated expression of VEGF, and that a COX-2 inhibitor would facilitate the management of conditions involving angiogenesis.
British Journal of Pharmacology 07/2000; 130(3):641-9. · 4.41 Impact Factor
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ABSTRACT: Previous studies on the rdw rat have suggested that its dwarfism is caused primarily by dysfunction of the thyroid gland. In this study, rat thyroid glands were analyzed endocrinologically and morphologically to clarify the primary cause of dwarfism in the rdw rat. The rdw rat showed lowered thyroid hormone (T4 and T3) levels but elevated TSH in serum. The rdw thyroid gland was almost proportional in size and it was not goiter in gross inspection. Our histological investigation produced three results that may lend important evidence in understanding the problem in the thyroid gland of rdw rats. First of all, secretory granules could not be detected in the follicular epithelial cells of the rdw. Secondly, thyroglobulin was found at very low levels in the follicular lumen by immunohistochemical analysis. In contrast, it could be detected in a substantial quantity inside the dilated rER and in the huge vacuoles that are formed by swelling of the rough endoplasmic reticulum (rER) at the basal side of the follicular epithelial cells. Additionally, the nucleus of the follicular epithelial cells was pressed to the luminal side by the enlarged rER. These morphological changes would indicate that the transport of thyroglobulin is stopped at or before the formation of the secretory granules and thyroglobulin is not secreted into the follicular lumen. The rdw characterization strongly supports that rdw dwarfism is induced by hypothyroidism due to some defect(s) in the thyroid gland.
The Anatomical Record 06/2000; 259(1):60-6.
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ABSTRACT: To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.
Journal of Electron Microscopy 02/2000; 49(3):445-51. · 1.31 Impact Factor
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ABSTRACT: We studied the distributions of laminin-5 and hemidesmosome components, HD1/plectin and BP230, in the submandibular glands of adult and developing mice. In adult mice, laminin-5 was expressed in the basement membranes of both the myoepithelial cells and excretory ducts. The former expression was predictable because laminin-5 is a ligand for hemidesmosomes, which appear in myoepithelial cells and stratified epithelium. However, the latter expression pattern suggested that the non-stratified epithelium of the excretory duct might also be associated with hemidesmosomes. During fetal development, laminin-5 was found in the basement membrane of developing ducts but not epithelial end buds in which future lobules are formed by epithelial branching. The expression of HD1/plectin but not BP230 was noted in the developing duct at early embryonic stages, indicating the presence of type II hemidesmosomes. Expression of BP230 appeared in the excretory duct epithelium at around the day of birth. At this stage, the typical hemidesmosome was observed in the duct epithelium. Our results suggest that laminin-5 is involved in duct development rather than epithelial branching. The results also suggest that the developing duct epithelium interacts with laminin-5 through the type II hemidesmosome, which later matures into a typical hemidesmosome upon the onset of expression of BP230.
Histochemie 01/2000; 112(6):417-25. · 2.59 Impact Factor
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ABSTRACT: The proliferation activity of component cells and its regional differences in the regenerating rat pancreas after 90% pancreatectomy were examined by bromodeoxyuridine (BrdU) immunohistochemistry. Cells of the ductal system and the centroacinar cells showed a rapid increase in labeling indices at day 2 after pancreatectomy, followed by a second peak of a mild increase at days 5 to 7. No regional difference in the labeling index was recognized in the ductal elements. In contrast, the labeling index of acinar cells started to increase at day 3, reaching a definite peak at day 5. Furthermore, acinar cells in the region close to the duodenum had labeling indices more than 2 times higher than those in the portions further away from the duodenum. Acinar cells increased in number as early as from day 3 after surgery. These result suggested that the parental cells of regeneration were located in the ductal epithelium. It is highly probable that the proliferation of acinar cells is controlled by some unknown trophic factor(s) which is released locally from the duodenum, but does not involve a neural or a circulatory route. The phenomenon may be closely linked to the known fact that the incidence of pancreatic cancer is highest in the head region.
Archives of Histology and Cytology 11/1999; 62(4):337-46. · 0.57 Impact Factor
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ABSTRACT: The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.
Archives of Histology and Cytology 11/1999; 62(4):347-54. · 0.57 Impact Factor
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S Yamashina
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ABSTRACT: The current curriculum reform is directed toward improvement of students' ability of clinical practice in primary care, and a substantial system of bedside learning is being extensively explored. Introduction of clinical clerkship necessarily forces qualitative changes in the teaching of basic medical sciences. A large number of Japanese medical schools is now attempting to establish a new educational system that makes it possible to change from "didactic education" by lectures and laboratory studies to "self learning education" by means of tutorials and other systems. In this context, Japanese anatomists are urgently requested to develop new and effective clinically oriented educational systems in teaching gross anatomy as a shift from the traditional teaching as a science of morphology. Evaluation of faculty staff's teaching achievement is about to start in many medical schools. Research activities can be evaluated quantitatively. In contrast, measurement of educational activities of faculty staff is very difficult, and all medical schools are devoted to construct effective and liable systems. Anatomy faculty is obliged to devote more time than that of other disciplines in education-related activities such as cadaver collection and associated business. How to evaluate adequately these invisible activities is an issue to be solved before the introduction of a self assessment system. Successful solutions to these issues are critical for production of future anatomists.
Anatomical Science International 09/1999; 74(4):491-3.
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ABSTRACT: High speed laser confocal microscopy (8 ms/image) was applied to the dissociated parotid acini as a model to study Ca2+ signaling mechanisms in non-excitable exocrine secretory cells. Immunofluorescence microscopy showed the localization of IP3 receptor type 2 along the apical membrane region. Muscarinic stimulation with carbachol evoked a rise in [Ca2+]i that was initiated from apical region and propagated into basal region as Ca2+ waves. This was most clearly observed when extracellular Ca2+ was omitted. Carbachol also triggered the abrupt increase of [Ca2+]i simultaneously at both basal and apical regions in many acini. Within an acinus, each cell responded synchronously. The present results suggest that one Ca2+ initiation site in the rat parotid acinar cell is apical region, corresponding to the localization of IP3 receptors. Another Ca2+ initiation site is basal region, which seems to be related to Ca2+ entry from extracellular medium and/or Ca2+ release from basally located organelles such as nuclei and endoplasmic reticulum.
Biochemical and Biophysical Research Communications 07/1999; 259(3):656-60. · 2.48 Impact Factor
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ABSTRACT: Mouse submandibular epithelium shows branching morphogenesis in mesenchyme-free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin-1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1-nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt-like matrix was formed on the epithelial surface in the LN1-nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV-including peptide (2097-2108), AG10 (2183-2194), AG32 (2370-2381) or AG73 (2719-2730) from the alpha1 chain, or YIGSR-including peptide (926-933) from the beta1 chain. Only AG73 (RKRLQVQLSIRT from the alpha1 chain carboxyl-terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1-nidogen can support the branching morphogenesis of submandibular epithelium even if LN1-nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.
Embryologia 05/1999; 41(2):207-16. · 2.21 Impact Factor
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ABSTRACT: Immunohistochemical localization of the epidermal growth factor receptor (EGFR) disclosed a wide distribution of this protein in diverse fetal tissues. This review focuses on some of the vast work on the expression and operation of the EGFR and its principal ligands in fetal organogenesis.
European Journal of Morphology 09/1998; 36 Suppl:92-7.
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ABSTRACT: To study the cell regulation mechanisms of human salivary secretion, surgical specimens of human parotid and submandibular glands were treated in vitro with isoproterenol (beta-agonist), carbachol (muscarinic agonist), and cytochalasin D (microfilament disruptive agent), and morphological changes occurring in serous acinar cells were observed. Control acinar cells treated without secretagogues exhibited only occasional examples of exocytosis. Microfilaments, revealed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) of F-actin fluorescence stained by rhodamine-phalloidin, were localized underneath the luminal membrane to separate the secretory granules from the luminal membrane. Following isoproterenol treatment, secretory granules made direct contact with the luminal membrane and many omega-shaped exocytotic profiles appeared. TEM and scanning electron microscopy (SEM) showed these profiles to be of granule size or somewhat smaller and to be provided on their cytoplasmic surface with coated pits. Furthermore, CLSM detected the appearance of F-actin fluorescence around the exocytosed granule membranes. Carbachol treatment also evoked the formation in acinar cells of omega-shaped exocytotic profiles some of which were larger than the granules and which exhibited neither coated pits nor associated F-actin fluorescence. To determine if microfilaments regulate the post-exocytotic process of membrane retrieval, we combined isoproterenol treatments with cytochalasin D or carbachol. Following these treatments, F-actin fluorescence surrounding the exocytosed membrane was dispersed or diffused and the exocytotic profiles enlarged remarkably. These results led to the hypothesis that exo/endocytotic processes in human salivary serous acinar cells are regulated differently under autonomic receptor control mediated by microfilaments.
European Journal of Morphology 09/1998; 36 Suppl:41-5.
