Review Application of Cationic Colloidal Gold Staining for the Demonstration of Anionic Sites
Kenji Saga
Department of Dermatology, Sapporo Medical University School of Medicine, Minami 1 Nishi 16, Chuo-ku, 060 Sapporo
Received for publication August 1, 1997 and in revised form September 12, 1997 and re-revised form October 20, 1997
Positively charged colloidal gold (cationic colloidal gold) stains negatively charged areas (anionic sites) on histological sections. Specimens embedded in hydrophilic resins are especially suitable for the precise localization of anionic sites with cationic gold in post-embedding method. Enzyme digestion on the sections embedded in hydrophilic resins before the staining with cationic gold enabled us to identify the molecules such as sialic acid, chondroitin sulfate, and heparan sulfate which cause anionic charge. Studies using cationic colloidal gold have been published from various fields of biology and medicine. In the kidney, glomerular basement membranes and glycocalyx of epithelial cells show anionic sites and these negative charges are thought to play important roles in the selective permeability in the glomerulus. Distributions of anionic sites in the glomerular basement membranes and epithelial cells are altered in diseases and in experimentally induced nephropathy. My laboratory has studied the distribution and enzyme susceptibility of anionic sites in human sweat glands, epidermis, and skin tumors. Cationic gold stains eccrine and apocrine sweat glands in a different manner. This would be helpful for an accurate diagnosis of sweat gland tumors. Data so far obtained suggest cationic colloidal gold is a useful tool to study structures and functions of cells and tissues with histochemical methods.
Influence of Extracellular Matrix on the Lipogenesis of Cultured White Fat Cells
Nobuhisa Yonemitsu 1,3, Fernando Bittinger1,2, Thomas Gerheld 1, Hajime Sugihara 3, Christian Mittermayer 1 and Charles James Kirkpatrick 1,2
1 Institute of Pathology, the Technical University (RWTH) of Aachen, Pauwelsstra{\ss}e 30, D-52057, Aachen, Germany, 2 Institute of Pathology, the Johannes Gutenberg University of Mainz, Langenbeck stra{\ss}e 1, D-55101, Mainz, Germany and 3 Department of Pathology, Saga Medical School, Nabeshima 5-1-1, Saga-city, 849-8501
Received for publication April 2, 1997 and in revised form October 1, 1997
Collagenase digests from subcutaneous fat tissue of new born rats were cultured on different types of collagen gel containing 50% type I collagen, on fibronectin (Fn) or on laminin (Lm). On day 6, 17% of cells on a plastic substratum differentiated and had multilocular or unilocular cytoplasmic lipid droplets (CLDs). Cells on each type of collagen and on Lm had more CLDs than those on a plastic substratum. The extent of lipogenesis showed the following decreasing order: cells on Lm (80%), on type IV+I collagen (70%), on type I collagen (52%), on type III+I collagen (36%), on type II+I collagen (32%). On day 14, most cells on Lm became unilocular fat cells. Cells on Fn showed delipidation and showed decreased reactivity to insulin stimulation.
Immunohistochemically differentiated fat cells were positively stained with anti-laminin or anti-type IV collagen antibodies. These results provide evidence that Lm and type IV collagen promote fat cell differentiation, whereas Fn facilitates dedifferentiation into the fibroblast-like fat cell.
Alkaline-phosphatase and Ca2+-ATPase Cytochemistry of 'Seamless' Endothelial Cells in the Rat Brain
Katsukuni Fujimoto
Department of Anatomy, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192
Received for publication May 13, 1997 and in revised form November 21, 1997
Capillaries having lumen formed by perforation of a single endothelial cell - the socalled seamless endothelial cell - have been reported by Wolff [30]. Seamless endothelial cells occur in the central nervous system and in other organs. This type of cell is more frequently observed where there is extensive vascularization. In the rat, the blood-brain barrier (BBB) is completed around the third week after birth. The activities of several enzymes correlate well with the functional heterogeneity of the microvascular endothelial lining. Alkaline phosphatase (ALPase) and Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) are representative enzymes revealing capillary permeability. In the present communication, the fine structural localizations of ALPase and Ca2+-ATPase were studied in the brain of young adult rats, with special
attention being directed towards the enzymatic activities of seamless endothelial cells.
Cytochemical Localization of Ca2+-, Mg2+-ATPase on the Limiting Membrane of Peroxisomes Isolated from Renal Tubules of Rats
Tutik Wresdiyati and Takashi Makita
Department of Veterinary Anatomy, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi City 753
Received for publication May 14, 1997 and in revised form October 9, 1997
The cytochemical localization of Ca2+-, Mg2+-ATPase in peroxisomes isolated from rat renal tubules was investigated at the ultrastructural level. The reaction products were localized mainly on the cytoplasmic
surface of the isolated peroxisomes, but also to some extent on the matrix side of the limiting membrane. Neither Ca2+- nor Mg2+ ATPase activity was affected by treatment with levamisole, an inhibitor of alkaline phosphatase. Ca2+-ATPase activity was inhibited by thapsigargin and myricetin, but not by PCMB. Mg2+-ATPase activity was inhibited by PCMB. It was inhibited slightly by thapsigargin, but was not affected by myricetin.
Postnatal Development of the Structure of the Peripheral Vestibular System of the Gerbil
Noriko Kawamura, Muneyuki Ito and Akiko Seto-Ohshima
Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-03
Received for publication June 25, 1997 and in revised form September 4, 1997 and re-revised form October 9, 1997
Postnatal development of the peripheral vestibular system and surrounding components in the gerbil was studied to obtain topological information for electrical vestibular stimulation. At postnatal day 1, the peripheral vestibular nerve had already reached the vestibular apparatus. The central vestibular nerve extended posteriorly to enter the brainstem at a sharp angle to anterior-posterior axis. During development, the vestibular ganglion was progressively displaced caudally, along with the vestibular nerve and the ventrally located facial nerve, increasing the angle to the anterior-posterior axis to nearly 90$\circ$ by postnatal day 30. Contact between the anterior part of the vestibular ganglion and the facial nerve, and that between the posterior part of the ganglion and the cochlear nerve were observed at all ages. These regions were considered unsuitable for electrical stimulation because of the high risk of stimulating the facial or cochlear nerve, similar to those regions in the adult. These results as well as the observation that the diameter of the lateral semicircular canal remained nearly constant showed that the technique we used for electrical vestibular stimulation in adult animals was apparently a good choice in younger animals as well.
Immunohistochemical Localization of Laminin, Collagen Type IV and Heparan Sulfate Proteoglycan in Human Colorectal Adenocarcinoma: Correlation with Local Invasive Pattern and Lymph Node Metastasis
1 Department of Oral Pathology, Faculty of Dentistry, Kyushu University 61, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 and 2 Department of Surgery, Chihaya Hospital, 2-30-1, Chihaya, Higashi-ku, Fukuoka 813-0044
Received for publication July 3, 1997 and in revised form October 1, 1997
The extent of immunohistochemical localization of such basement membrane components (BMs) as laminin (LN), collagen type IV (C-IV) and heparan sulfate proteoglycan (HSPG) were investigated in 78 samples of human colorectal carcinoma, in correlation with histology of the invasive pattern at the parenchymal-stromal border of the invasive margin (PSB) and lymph node metastasis. The immunostaining ratio of BMs at the PSB was significantly lower in the type IV invasive pattern (PSB-IV) than in the other types, and was also significantly related to the incidence of lymphatic invasion and lymph node metastasis. A close relationship was also demonstrated between the PSB-IV and the prognosis of the patient or recurrence of the carcinoma, although there was no statistical significance. Furthermore, the immunostaining ratios of the LN and C-IV in the central areas (CAs) of the carcinoma tissue were also significantly lower in samples with lymph node metastasis than those without metastatic focus. These results thus indicate that the lower degree of immunostaining ratio of BMs at the PSB in human colorectal carcinomas is closely related to both the lymph node metastasis and the patient prognosis, while in addition the varied localization of BMs at the PSB affects diverse histopathological features in the invasive margin of carcinoma tissue. Moreover, the decreased immunostaining deposition of BMs in the CAs of carcinoma tissue also plays an important role in the pathway of lymph node metastasis. Therefore, further attention should be paid to the cellular biology of carcinoma cells localized in the CA, as well as those localized at the PSB, in order to disclose the metastatic cascade of colorectal carcinoma.
Key words: Human colorectal carcinoma, Basement membrane components, Invasive pattern, Immunohistochemistry, Lymph node metastasis and prognosis
1 Faculty of Health Science, Kobe University School of Medicine, 7-10-2, Tomogaoka, Sumaku, Kobe, 654-0142, 2 Miki City Hospital, 58-1, Kasa, Miki, Hyogo, 673-0402 and 3 Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuoku, Kobe, 650-0017
Received for publication July 24, 1997 and in revised form September 12, 1997 and in re-revised form November 21, 1997
The depostion of lipoprotein(a) (Lp(a)) of coronary sclerosis was immunohistochemically studied by using 9 autopsy cases and compared with the infiltration of the inflammatory cells such as macrophages (Mp), T-lymphocytes (T-cells) and B-lymphocytes. Lp(a) deposition was recognized in 6 cases out of 9. The type of atherosclerosis was classified according to the classification of the American Heart Association. In the initial lesions, type I, Lp(a) was deposited unevenly in the subintimal region and the infiltration of Mp was recognized without lymphocytes. In the advanced lesion, type III, Lp(a) was deposited primarily among extracellular tissues. In the more advanced lesion, type IV, Lp(a) was identified within Mp as well. In the shoulder region of the advanced lesion, the Lp(a) deposition and the infiltration of Mp and T-cells were recognized to colocalize in the limited zone without smooth muscle cells. This finding suggested this limited zone would be vulnerable to stress and induce the following rupture
of the lipid cor. In addition, Lp(a) was deposited and formed double layers in the advanced sclerotic fibrous lesion. These pathological findings suggested the importance of Lp(a) in atherosclerosis and the subsequent rupture of the advanced lesion in relation to inflammatory cells.
Expression of Vascular Endothelial Growth Factor and Its Receptor flk-1 in Soft Tissue Sarcomas: Correlation to Cathepsin Expression and Prognosis
Peter W\"{u}rl 1*, Helge Taubert 2*, Axel Meye 2, Dieter Berger 2, Christine Lautenschl\"{a}ger 3, Hannelore Schmidt 2, Raoul Hinze 2, Friedrich-Wilhelm Rath 2 and Manfred Sch\"{o}nfelder 1
1 Surgical Clinic 1, University Leipzig, Liebigstrasse 20a, D-04103 Leipzig, Germany, 2 Institute of Pathology, Martin Luther University Halle, Magdeburger Strasse 14, D-06097 Halle/Saale, Germany and 3 Institute of Medical Biometry and Informatics, Martin Luther University Halle, Kraussenstrasse 14, D-06097 Halle/Saale, Germany
Received for publication July 25, 1997 and in revised form January 19, 1998
Angiogenesis and decomposition of the extracellular matrix are essential suppositions for the growth of solid tumors. Cathepsins are a class of proteinases able to degradate extracellular matrix. They may have significance as a prognostic factor in soft tissue sarcomas (STS). Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen and inducer of angiogenesis in normal as well as in tumor tissue. Little is known about VEGF and possible correlations to cathepsins in STS. Therefore we investigated the expression of VEGF and the VEGF-receptor flk-1 in 36 STS with known expression rates for cathepsin L, S, D and procathepsin L. Twelve samples (33.3%) were positive for VEGF, and 20 (55.6%) for flk-1. This is comparable with the known expression rates in carcinomas. We have found neither a significant correlation to histomorphological and clinical parameters nor to cathepsin expression. Summarizing, we could not prove any prognostical relevance for immunohistochemical detection of VEGF or flk-1. A correlation between cathepsin expression and the investigated angiogenic factors was also not evident.
1 Department of Pathology II, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka 570-8506 and 2 Department of Obstetrics and Gynecology, Kurume University School of Medicine, 67 Asahi, Kurume, Fukuoka 830-0011
Received for publication September 16, 1997
The mechanisms of gliomatosis peritonei, composed exclusively of mature glial tissue implants on the peritoneum or omentum, are not yet understood. In the present study, the expressions of metalloproteinases (MMPs; MMP-1, -2, -3, and -9) and tissue inhibitors of metalloproteinases (TIMPs; TIMP-1 and -2) were examined in ovarian immature teratomas with and without glial implants to evaluate the roles of MMPs and TIMPs in gliomatosis peritonei. The immunostaining showed a high expression of MMP-1 in the glial fibrillary acidic protein (GFAP)-positive cells in the glial implants, and in the primary immature teratomas regardless of the existence of gliomatosis peritonei. A weak expression of MMP-2 was seen in both primary and implant sites in the case with gliomatosis peritonei, but not in the cases without glial implants. In contrast, a high TIMP-2 expression was seen in the glial cells in the cases without gliomatosis peritonei but was absent in the primary as well as peritoneal implants in the case with gliomatosis peritonei. The expressions of MMP-3, MMP-9, and TIMP-1 were universally negative. These results suggest that the loss of TIMP-2 plays a crucial role in the development of glial implants of ovarian immature teratoma.
Platelet-type Arachidonate 12-Lipoxygenase in Mouse Gastrointestinal Tract
Michihiro Nakamura, Shozo Yamamoto and Kazunori Ishimura*
Department of Biochemistry, Tokushima University, School of Medicine, Kuramoto-cho, Tokushima 770 and *Department of Anatomy, Tokushima University, School of Medicine, Kuramoto-cho, Tokushima 770
Received for publication October 1, 1997
12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid, and produces 12S-hydroperoxy-arachidonic acid. When mouse gastrointestinal tract was immunostained with an antiserum against human platelet 12-lipoxygenase and examined by light microscopy, positively-stained cells were found in the epithelium of stomach and small and large intestines. Electron microscopic immunohistochemistry revealed that the positively-stained cells were open-type enteroendocrine cells. The endocrine cells had many granules of heterogeneous sizes, forms and internal structures, and most of the granules had electron lucent area. 12-Lipoxygenase was localized in the cytoplasm, but not in the nucleus, plasma membranes and other subcellular organelles. Double immunostaining revealed that all 12-lipoxygenasepositive cells contained serotonin. With an antiserum against 12-lipoxygenase isozyme of porcine leukocytes, no positively-stained cells were observed in gastrointestinal epithelium.
Note Cytochemical Studies on Carbohydrates in the Reticular Cells of the Rat Splenic Cord
Hiromi Ueda and Osamu Fujimori
Department of Anatomy, Nagoya City University Medical School, Nagoya 467-8601
Received for publication May 6, 1997 and in revised form January 22, 1998
Carbohydrates in the reticular cells of the rat splenic cord were studied by means of a series of electron microscopic cytochemical methods. The results indicated that the reticular cells contained an appreciable abundance of glycogen particles in the cytoplasm. In the free surface coat of the plasma membrane, the cells included neutral glycoprotein, which were devoid of detectable amounts of acidic glycoconjugates. The possible physiological significance of these carbohydrates is discussed, with special reference to their characteristic cytochemical features examined in the present study.
Key words: Electron microscopy, Cytochemistry, Carbohydrates, Reticular cells, Splenic cord