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Volume 36 (2003)
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- Issue 4 Pages 249-
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Volume 36, Issue 4
Displaying 1-20 of 20 articles from this issue
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REVIEW
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Application of Genetic Engineering Technologies for the Study of Pituitary Development and NeoplasmsRobert Yoshiyuki Osamura, Shunsuke Miyai, Noboru Egashira, Susumu Take ...Article type:
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2003 Volume 36 Issue 4 Pages 249-254
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSOur studies and review of the literature reported by other investigators indicate the essential use of genetic engineering technology to study the physiologic and pathologic mechanisms of transcription of hormone production and the mechanisms of secretion of appropriate bioactive peptides from the prohormones in the endocrine cells which are equipped with secretory granules.
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Kazushige Ogawa, Masashi Tsuji, Shingo Tsuyama, Fumihiko SasakiArticle type:
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2003 Volume 36 Issue 4 Pages 255-262
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSWe previously found that parietal cells in guinea pigs starved for a few days contained giant mitochondria while the cells of animals starved and then injected with histamine mostly did not. To test whether mitochondria change their size and activity according to energy demands for acid secretion, we examined the dynamics of mitochondria stained by rhodamine 123 in living parietal cells in gastric glands under a confocal microscope. The glands were isolated from guinea pigs starved for 60-72 hr. Because mitochondria were closely packed in the cytoplasm, we failed to observe the morphological changes of each mitochondrion in parietal cells. However, we successfully observed that 10-5 M histamine induced an increase in the fluorescence intensity (the concentration of rhodamine 123) in mitochondria in subpopulations of parietal cells; the fluorescence intensity increased sharply within minutes after the histamine administration in some cells, while it gradually increased from just after the administration in other cells. We also found that a subpopulation of mitochondria within a parietal cell responded to the secretagogue. The findings suggest that parietal cells exist as a heterogeneous population in gastric glands and contain heterogeneous mitochondria in terms of their mitochondrial response to histamine.
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Airo Tsubura, Katsuhiko Yoshizawa, Katsuji Kiuchi, Kaei MoriguchiArticle type:
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2003 Volume 36 Issue 4 Pages 263-270
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSRetinitis pigmentosa (RP) is a human disease characterized by loss of photoreceptor cells leading to visual disturbance and eventually to blindness. Establishment of reliable animal models is essential for better understanding of this disease and approaches to therapeutic intervention. N-Methyl-N-nitrosourea (MNU)-induced retinal degeneration is highly reproducible, and involves photoreceptor cell loss that ends approximately 7 days after a single systemic administration of MNU. Although the triggering mechanisms of pathogenesis are different and the apoptosis cascade may differ from human RP, MNU-induced photoreceptor cell loss is due to apoptosis. Here, we describe disease progression and therapeutic trials of MNU-induced retinal degeneration in animals.
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Yutaka Tsutsumi, Shingo KamoshidaArticle type:
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2003 Volume 36 Issue 4 Pages 271-280
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSFor investigating the biological significance of apoptosis, the exact and sensitive histochemical identification of apoptosis and apoptotic cells is essential. However, we need to recognize both the pitfalls and caveats in performing histochemical staining and in interpreting the findings obtained. DNA fragmentation-based approaches, such as TdT-mediated dUTP-biotin nick end-labeling (TUNEL), in situ nick translation (ISNT) and immunostaining for single-stranded DNA, represent DNA alterations in the apoptotic cell, but they are technically unstable and occasionally give false positive and false negative findings. In contrast, immunostaining for intracellular proteins cleaved and activated by caspases, including cleaved caspase 3, cleaved poly(ADP-ribose) polymerase, cleaved cytokeratin 18 and cleaved actin (fractin), is technically reproducible, but the intracellular accumulation of the activated proteins is not necessarily synchronized. The present review focuses on the pretreatments for enhancing the sensitivity of these techniques, as well as their limitations and comparisons in histochemically demonstrating apoptosis and apoptotic cells.
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Susumu Takekoshi, Hidetaka Nagata, Kentaro Matsuzaki, R. Yoshiyuki Osa ...Article type:
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2003 Volume 36 Issue 4 Pages 281-285
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSNumerous studies have shown that lipid peroxidation is involved in the pathogenesis of various degenerative diseases including atherosclerosis, diabetes, cancer, inflammation, drug-induced toxicity, and neurodegenerative disease. Protein kinase C (PKC), a lipid activated kinase, is a key enzyme for intracellular signaling pathway in physiological conditions. Recently considerable evidence has accumulated to confirm that lipid peroxidation participates in intracellular signal transduction in the pathophysiology of aerobic organisms. Lipid peroxides, such as hydroperoxy fatty acids, oxidized cholesteryl linoleates and 4-hydroxy-2-nonenal, efficiently stimulate PKC in the cells. More importantly, oxidized diacylglycerol strongly stimulates PKC as much as phorbol ester, a strong tumor-promoting PKC activator. This review article describes PKC signaling pathway, the role of lipid peroxides in the intracellular signaling, the significance of lipid peroxidation in pathophysiology, and the cellular defense systems against oxidative stress.
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Hiroshi Nagura, Yoko Nagura, Shin Fukudo, Hironobu SasanoArticle type:
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2003 Volume 36 Issue 4 Pages 287-292
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSMutual communication among the immune, neuroendocrine and metabolic systems is essential for the maintenance of gastrointestinal mucosal function. A unique barrier system by the mucosal immune system handles a myriad of infectious and food antigens, while the neuroendocrine system interplays with the immune system in the intestinal mucosa. The close relation between these two systems is associated with the pronounced effects of stress and metabolic changes.
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Ronald A. DeLellis, Sandra J. ShinArticle type:
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2003 Volume 36 Issue 4 Pages 293-298
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSImmunohistochemistry has played a major role in the practice of surgical pathology over the past three decades. These techniques have been of particular value in the diagnosis and classification of tumors of the adrenal cortex. The intermediate filament phenotype of adrenocortical carcinomas includes positivity for vimentin (80-100%), cytokeratin (40-50%) and neurofilament proteins (50%). Other markers that have proven useful for the diagnosis of these tumors include melan A (A103), alpha inhibin, synaptophysin, calretinin, D11 and the nuclear protein adrenal 4 binding protein. The judicious use of these markers permits the distinction of adrenocortical carcinomas from other tumor types and may also be of value in the identification of the functional characteristics of these neoplasms.
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Tetsuji NagataArticle type:
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2003 Volume 36 Issue 4 Pages 299-315
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSX-ray microanalysis is a useful technique to qualify and quantify basic elements in biological specimens. This article reviews the principles and techniques for applying intermediate high voltage electron microscopy to X-ray microanalysis of various elements in biological specimens developed in our laboratory since the late 20th century. We first quantified the endproducts of histochemical reactions such as Ag in radioautographs, Ce in acid phosphatase reaction and Au in colloidal gold immunostaining using semi-thin sections by high voltage electron microscopy at 300-400 kV. We then analyzed various trace elements such as Zn, Ca, and S, which originally existed in the cytoplasmic matrix or cell organelles of various cells in different organs, and some absorbed elements introduced by experimental administration into cells and tissues such as Al, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning, freeze-drying, or freeze-substitution. As a result, we showed that the peak to background (P/B) ratios of all the elements analyzed had high P/B ratios at 300-400 kV. It was concluded that X-ray microanalysis using semi-thin sections by high voltage electron microscopy is of great utility for quantifying trace elements in biological specimens.
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TECHNICAL ADVANCEMENT
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Kuniaki Takata, Yukiko Takahashi, Toshiyuki Matsuzaki, Yuki Tajika, Ta ...Article type:
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2003 Volume 36 Issue 4 Pages 317-323
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSExpression of genes followed by the examination of the localization of their products by histochemical methods is one of important and powerful approaches to elucidate the role of genes of interest. We show here a simple and versatile electroporation method for the introduction of plasmids into cells cultured on coverslips for histochemical examination. Electroporation apparatus was assembled using a 35-mm plastic dish and aluminum wire. The apparatus was placed over the coverslip and plasmid DNA was applied onto it. Green fluorescent protein (GFP) was used as a reporter of transfection. Under the appropriate settings of voltage, duration, and pulse numbers, successful transfection was easily achieved in 3T3-L1 fibroblasts, HeLa cells, Cos7 cells, and MDCK cells. The electroporation method described here is simple, easy, rapid, and economical, and may be best suited for the screening of many genes for their histochemical examination.
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Shin-ichi Izumi, Masashi Shin, Kunio Takano, Paul K. Nakane, Takehiko ...Article type:
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2003 Volume 36 Issue 4 Pages 325-330
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSIn situ hybridization has been widely used for localization of specific genes in interphase cell nuclei. The sites of signals, however, do not represent the activity of the gene transcription in the nuclei. To differentially localize genes in active and inactive states, the effect of nuclease digestion prior to in situ hybridization using PCR products of DNA fragments from human X-chromosome, as a probe DNA, was examined in female neutrophils. According to the conventional protocol for chromosomal in situ hybridization, we detected two dot signals in the nucleus. However, the use of Ca/Mg-dependent endonuclease, which extracts inactive gene DNA, resulted in the loss of the one signal of the outer region of the nucleus, but not that of the inner one. In contrast, when exogenous DNase I, which extracts active gene DNA, was used in place of Ca/Mg-dependent endonuclease, the inner signal, but not the outer one, was almost lost in neutrophils. Considering that the outer signal represents inactive X-chromosome DNA in the site of female neutrophil nuclei, these results indicate that the use of our modified protocol may enable us to discriminate the signals of active and inactive genes in interphase cells by in situ hybridization.
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REGULAR ARTICLE
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Sumio NishikawaArticle type:
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2003 Volume 36 Issue 4 Pages 331-334
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSSarcoplasmic or endoplasmic reticulum Ca2+ ATPase (SERCA2) was localized in the ameloblasts of rat incisors by immunohistochemistry. Secretion ameloblasts and maturation ameloblasts, including both ruffle-ended and smooth-ended cells, were labeled with anti-SERCA2 antibodies, with maturation ameloblasts showing stronger binding than secretion ameloblasts. The pattern of labeling was cytoplasmic and diffuse. Papillary layer cells were not labeled with anti-SERCA2 antibodies. These results suggest that the SERCA2 in ameloblasts is intimately involved in mineralization during amelogenesis.
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Masanori Murakoshi, R. Yoshiyuki Osamura, Keiichi WatanabeArticle type:
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2003 Volume 36 Issue 4 Pages 335-343
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSIn order to confirm the relationship between glutathione-peroxidase (GSH-PO) and Bcl-2, we have studied the immunocytochemical localization of GSH-PO, Bcl-2, androgen receptor (AR) and apoptosis in the rat ventral prostatic cells in the presence and absence of testosterone. Male Crj:CD(SD) IGS rats were divided into four experimental groups. Group 1 consisted of untreated controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for 3 or 7 days after two days of castration, respectively. In group 1, GSH-PO was predominantly observed in the glandular epithelial cells. Bcl-2 was localized exclusively in the cytoplasm of the glandular epithelial cells. AR was localized in the nuclei of the glandular epithelial cells. In group 2, the intensity of the staining of GSH-PO, Bcl-2 and AR markedly decreased. However, the number of apoptotic cells (apoptotic index) markedly increased. In groups 3 and 4, the immunoreactivities of GSH-PO, Bcl-2 and AR clearly recovered. These findings strongly suggest that the expression of GSH-PO and Bcl-2 in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent. Furthermore, co-expression of GSH-PO and Bcl-2 in the prostatic cells are considered to be normal or adaptive aspects of the cells.
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Kazuhisa Hasui, Atae Utsunomiya, Shuji Izumo, Masamichi Goto, Suguru Y ...Article type:
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2003 Volume 36 Issue 4 Pages 345-352
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSHuman T-cell lymphotropic virus type 1 (HTLV-1) proviral DNA p40Tax (Tax) protein plays an important role in adult T-cell leukemia/lymphoma (ATLL) oncogenesis, but it has yet to be determined whether or not ATLL leukemic cells express Tax. In a study designed to answer this question, we prepared peripheral blood tissue specimens (PBTS) from four acute and seven chronic ATLL cases and three B-cell chronic lymphocyte leukemia (B-CLL) cases, and examined their immunological phenotype and Ki67 labeling index (Ki67LI) by means of antigen-retrieval immunohistochemistry. In three of the chronic ATLL cases, the difference in Ki67LI between leukemic cells that underwent either 3 hr, or more than 6 hr of sedimentation and coagulation while preparing PBTS suggested that the Ki67LI in PBTS represents reproliferating cells. Tax expression was examined by means of the ImmunoMax method. In two acute and three chronic type ATLL cases, leukemic cells expressed Tax in parallel with the Ki67LI, suggesting that Tax expression was related to reproliferation in PBTS. Leukemic cells in one case each of acute and chronic type ATLL did not express Tax. In one case of acute ATLL after chemotherapy, some cells expressed Tax among the other cells. These results indicate that ATLL leukemic cells are able to express an extremely small amount of Tax during reproliferation.
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Mika Iwami, Ikuo Tooyama, Ayae Kinoshita, Akinori Matsuo, Yutaka Oomur ...Article type:
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2003 Volume 36 Issue 4 Pages 353-359
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSRecent studies have shown that FGF-1 (aFGF) and FGF-2 (bFGF) are expressed in the adrenal glands. In order to understand the roles of FGFs in the adrenal gland, we investigated the expression of the fibroblast growth factor receptor-1 (FGFR1) in rat adrenal gland using reverse transcription-polymerase chain reaction (RT-PCR) method, Western blot and immunohistochemistry. RT-PCR experiment using a primer set to amplify the acidic region of FGFR1 gave two bands of about 426 bp and 160 bp in both the adrenal cortex and medulla. The sizes of the large and small PCR products corresponded well to the three IgG-like domain (long form) and two IgG-like domain (short, or secreted form) FGFR1, respectively. Western blotting analysis using an antibody capable of detecting the acidic region of FGFR1, gave two bands in the adrenal gland with molecular weights of about 130 kDa and 75 kDa, corresponding to the long and the secreted form of FGFR1, respectively. Immunohistochemical study using the antibody demonstrated that FGFR1-positive cells were distributed mainly in the zona glomerulosa of the adrenal cortex and medulla. A few positive cells were scattered in the zona fasciculata and reticularis. In the adrenal medulla, almost all cells were positive for FGFR1. Double immunostaining revealed that FGFR1 colocalized with tyrosine hydroxylase-immunoreactive catecholamine cells in the adrenal medulla.
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Shinobu Umemura, Johbu Itoh, Susumu Takekoshi, Hideaki Hasegawa, Masan ...Article type:
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2003 Volume 36 Issue 4 Pages 361-366
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSWe occasionally encounter decreased staining of methyl green (MG) in heated sections for antigen retrieval. To examine the relation between MG staining and conditions of DNA strands, DNA in various conditions were blotted on the transfer membrane and stained. It was found that staining intensity of MG was changed due to structural alterations of DNA strands. Double stranded DNA in various solutions, denatured single-stranded DNA, DNase treated samples, and short length single-stranded oligonucleotides were examined. DNA diluted in high concentrated SSC was stained more strongly than low concentrated SSC or distilled water. Denatured and/or DNase treated DNA showed decreased staining. It was especially noteworthy that oligonucleotide DNA strands of poly C and poly G strands were positively stained, but very faint MG staining was also observed for poly A while no staining was noted for poly T. In conclusion, we presented that MG staining intensity was altered by the conditions of DNA in vitro. Intense MG staining for basic acids with three hydrogen bonds, cytosine and guanine, suggest a possible mechanism for MG staining.
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Johbu Itoh, Shinobu Umemura, Hideaki Hasegawa, Masanori Yasuda, Susumu ...Article type:
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2003 Volume 36 Issue 4 Pages 367-376
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSObservations of the fine structures of subcellular organelles and their pathophysiological changes have been made by many investigators by utilizing the many advantageous functions of confocal laser scanning microscopy (CLSM). However, reports of CLSM observations of fine inner structural changes of nuclei are rather rare.
The usefulness and value of counterstaining in histochemical staining is widely appreciated. While observing methyl green (MG) nuclear-counterstained sections by CLSM, we found that fluorescence emitted through MG was clearly detected by CLSM. It is generally accepted that MG reacts specifically to double stranded (dbs)-DNA, as was proved by Umemura [30] in our laboratory.
In the present study, MG-stained nuclei of the absorptive cells (terminal differentiating cells) of rat intestinal villi were observed by CLSM, and changes in the intensity and distribution of MG staining were examined. The small intestinal epithelial cells were also found to be slowly undergoing apoptosis. The apoptosis is thought to occur during the process of terminal differentiation, which takes about 48 hr. To examine those apoptotic changes, a TUNEL method, in which the 3'ends of fragmented dbs-DNAs are labeled with histochemically detectable dUTP, was employed. The interrelationship between cell functions, which can be detected by various histochemical observations of cytoplasmic substances, and cellular differentiation state, can be elucidated by using MG in CLSM.
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Miyuki Yamamoto, Shoichi IsekiArticle type:
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2003 Volume 36 Issue 4 Pages 377-383
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSThe expression and localization of NGF and its high-affinity receptor, TrkA, in the rat carotid body was investigated by use of in situ hybridization and immunohistochemistry, respectively. The immunoreactivity for TrkA was detected in most of chief cells and sustentacular cells, as well as in some nerve axons and perineurial cells, but not in Schwann cells. In electron microscopy, the immunoreactivity was localized to the cytoplasm. The mRNA for NGF was localized to most of chief cells and possibly also to sustentacular cells. These results demonstrated that carotid body chief cells, having the neural crest origin, produce both NGF and TrkA, implying a paracrine-autocrine role of NGF in the carotid body.
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Hirokazu Ohtaki, Sakura Endo, Tomoya Nakamachi, Li Yin, Kenji Dohi, Yo ...Article type:
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2003 Volume 36 Issue 4 Pages 385-391
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSMolecular mediators such as intercellular adhesion molecule-1 (ICAM-1) have been implicated in the induction of neuronal damage after ischemia/reperfusion. However, the time-dependent expression of ICAM-1 after transient ischemia and the relative influence of ICAM-1 on neuronal cell death are not well understood. We performed immunostaining for ICAM-1 and apoptotic-like neuronal cell death by in situ by terminal UTP-nucleotide 3'-OH-DNA end labeling (TUNEL) after a 1-hr transient middle cerebral artery occlusion (tMCAO) in mouse. ICAM-1-like immunoreactivity (ICAM-1-ir) was detected only to a slight extent in the brains of sham-operated control. ICAM-1-ir after tMCAO was noted in the ischemic region of the ipsilateral hemisphere within 3 to 6 hr, and increased significantly from 24 to 96 hr. The ICAM-1-ir was mainly localized in the endothelium of blood vessels, and was also observed in astrocytes 24 hr after tMCAO. While the endothelial expression of ICAM-1-ir overlapped with that of TUNEL staining, the astroglial expression of ICAM-1-ir was observed around the periphery of infarction, which did not recognize TUNEL-positive reaction. These results suggest that ICAM-1 expression in the mouse brain increases after tMCAO, and that the endothelial expression of ICAM-1 could be indicative of the induction of neuronal damage via leukocyte invasion.
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Setsuya Fujita, Yuko YasudaArticle type:
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2003 Volume 36 Issue 4 Pages 393-398
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSThe elevator movement of the matrix cell is synchronous with the mitotic cycle. Its mechanism has been thought to be due to cyclic changes in cell adhesion to the neighboring cells and the basement membrane. The present study aims to analyze the cytoplasmic changes by means of immunohistochemical investigations of the distributions of the adhesion molecules, N-cadherin, α-catenin, β-catenin, F-actin, etc. It was found that the adherent junctions and junctions to ECM of the matrix cell disintegrate during mitosis, releasing the cell from the intercellular adhesions and the basement membrane. As a consequence, the mitotic matrix cell is rounded out and attracted to the ventricular surface where the junctional complexes remain unchanged during mitosis. After division, the daughter matrix cells recover their adhesion apparatuses and, extending their cytoplasmic processes, resume their downward elevator movement.
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Nobuyuki Karasawa, Terumi Takeuchi, Keiki Yamada, Mineo Iwasa, Genzoh ...Article type:
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2003 Volume 36 Issue 4 Pages 399-407
Published: 2003
Released on J-STAGE: November 18, 2003
JOURNAL FREE ACCESSWe determined the distribution pattern of cholinergic neurons in laboratory shrews (Suncus murinus) based on comparative morphology by an immunohistochemical method using an anticholine acetyltransferase (ChAT) antibody. ChAT-positive neurons were observed in the striatum, magnocellular nucleus and basal nucleus, pontine tegmentum and cranial nerve motor nuclei (IV, V, VII, X and XII). Specific findings were as follows: many ChAT-positive neurons were observed in the locus ceruleus (LC) and raphe dorsalis (RD), and the colocalization of ChAT-positive neurons and tyrosine hydroxylase in the LC and that of ChAT-positive neurons and serotonin in the RD were clarified. On the other hand, in the rat brain, ChAT-positive neurons were present in the central amygdaloid nucleus; however, this was not observed in the brains of laboratory shrews. Laboratory shrews are among the most primitive mammalian species and have a different distribution pattern of aminergic neurons, other than cholinergic neurons, from that in rats. A unique distribution pattern of ChAT-positive neurons in the laboratory shrew brain was thus noted.
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