Review Efficiency of Primed In Situ Labeling (PRINS) Method for Interphasic Chromosomal Screening: a Review
Franck Pellestor, Anne Girardet and Brigitte Andr\'{e}o
CNRS UPR 1142, 141 rue de la Cardonille, F-34396 Montpellier cedex 5, France
Received for publication May 6, 1998 and in revised form August 31, 1998
The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis. One of these techniques is the primed in situ labeling (PRINS) procedure which constitutes a fast and efficient alternative to conventional fluorescence in situ hybridization (FISH) for chromosomal detection. Based on the use of chromosome-specific primers, the PRINS method combines the high sensitivity of the PCR reaction with the cytological localization of DNA sequences. Using this approach, numerous cytogenetic applications have been successfully developed on various types of cells. With the development of rapid protocols producing reliable and reproductible results, the PRINS procedure has an enormous potential for cytogenetic investigations.
Key words: PRINS, In situ labeling, Aneuploidy, Interphase
Review Immunohistochemical Classification of Skeletal Muscle Fibers
Aiko Hori 1, Akihiko Ishihara 2, Shigeo Kobayashi 1 and Yasuhiko Ibata 3
1 Department of Bio-informatics, Graduate School of Informatics, 2 Laboratory of Neurochemistry, Faculty of Integrated Human Studies, Kyoto University, Kyoto 606-8501 and 3 Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto 602-0841
Received for publication May 11, 1998 and in revised form September 7, 1998
Mammalian skeletal muscle fibers have several types of myosin heavy chain (MHC) isoforms. Immunohistochemical techniques enable us to identify the MHC isoform type in a single muscle fiber, i.e., MHCI, MHCIIa, MHCIIx, or MHCIIb. In addition, hybrid single fibers having more than one MHC isoform type can be classified using immunohistochemistry. The presence of different MHC isoform(s) in a single fiber correlates with the maximal contractile velocity and the enzyme histochemical reaction of the myofibrillar adenosine triphosphatase. The adaptive response of the skeletal muscle is the basis of the fiber-type transformation depending on the phenotypic expression pattern in several MHC isoforms. In this review article, we address the availability of the monoclonal antibodies which recognize specific MHC isoform(s) in a single fiber.
Prothymosin Alpha Location in the Proliferative Compartments of the Digestive Tract Epithelium
Rosal\'{i}a Gallego 1, Tomas Garcia-Caballero 1, Elena Roson 1, Maximo Fraga 2, Fernando Dominguez 3 and Andres Beiras 1
1 Departments of Morphological Sciences, 2 Pathology and 3 Physiology, Faculty of Medicine, Santiago de Compostela, Spain
Received for publication October 20, 1997 and in revised form December 16, 1997 and re-revised form August 7, 1998
Prothymosin alpha is a polypeptide whose function is still a matter of debate. The present paper represents the first morphological description of the prothymosin alpha distribution in the human and rat digestive tract tissues using immunohistochemical techniques. In these epithelia prothymosin alpha immunostaining was localized in the nuclei of the proliferating cells, e.g. in the basal layer cells of the oesophagus, cells of the isthmus and the neck of the gastric glands, and crypt cells in the small and large intestines. At the ultrastructural level, prothymosin alpha immunolabelling was evident in the nucleus, being confined to euchromatin. The present data indicates that prothymosin alpha expression in the digestive tract epithelia is associated with proliferating cell populations. In agreement with this hypothesis, we found that the patterns of distribution of prothymosin alpha and proliferating cell nuclear antigen were similar. The distribution of prothymosin immunoreactivity was also similar to that obtained for bromodeoxyuridine when the animal was sacrificed 12 hr after injection. The finding that intracellular distribution of the prothymosin alpha in the digestive tract epithelia is identical to that found in other tissues supports the idea that prothymosin alpha must play a universal role in proliferating cells.
Subcellular Localization of Steroid Hormone Receptors during Mitosis
Shuji Yamashita
Keio Junior College of Nursing, Shinjuku, Tokyo 160-8582
Received for publication March 5, 1998 and in revised form October 5, 1998
The subcellular distribution of steroid hormone receptors (SHRs), estrogen receptors (ERs), progesterone receptors (PRs) and glucocorticoid receptors (GRs) in mitotic cells was examined using immunohistochemical techniques. ERs and PRs were localized in the uterus of mice and rats, respectively; the ovariectomized adult rodents were sacrificed 18-23 hr after17$\beta$-estradiol injection. GRs were demonstrated in the regenerating rat liver following partial hepatectomy. ERs and PRs were associated mainly with chromatin/chromosomes through all stages of the cell cycle in the uterine epithelium. Immunoreactivity was observed on the surface of mitotic chromosomes. GRs were localized exclusively in the nucleus of rat hepatocytes in interphase and were diffusely spread in the cytoplasm during mitosis, having dissociated from the chromosomes. GR immunoreactivity was recognizable frequently on the mitotic spindles. The distribution patterns of these SHRs seemed to be independent of hormonal status.
Demonstration of Biological Aggressiveness of Bone Giant Cell Tumor by the Comparative Study of Immunohistochemical Detection of DNA-instability and Cortical Bone Destruction by CT
Department of Orthopaedic Surgery, School of Medicine, Fukui Medical University, Shimoaizuki 23, Matsuoka, Fukui 910-1193 and * Department of Pathology, School of Medicine, Fukui Medical University, Shimoaizuki 23, Matsuoka, Fukui 910-1193
Received for publication May 19, 1998 and in revised form July 29, 1998 and re-revised form August 27, 1998
The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to benign (4 osteochondroma and 4 enchondroma cases), border-line (23 bone giant cell tumor, BGCT cases), and malignant (6 chondrosarcoma and 6 osteosarcoma cases) neoplastic lesions. The expression of tumor oncogene, c-myc was detected immunohistochemically. Proliferative activity was evaluated by PCNA-immunohistochemistry, and the quantitative analysis of the number, mean area, mean total area, the largest area, and maximum shape-irregularity of AgNORs in a nucleus were performed for all these cases. The results for 19 BGCT (82.6%) cases, 6 chondrosarcoma cases (100%), and 6 osteosarcoma cases (100%) were positive with the DNA-instability test, indicating their malignancy. All benign tumor cases were negative with the DNA-instability test. Reflecting the malignant character, all chondrosarcoma cases, all osteosarcoma cases and the BGCT cases positive with the DNA-instability test showed statistically higher values of PCNA-index; and all AgNORsparameters in comparison to those for benign tumor cases, and c-myc was positive for 66.7%, and 26.3%, of them, respectively. But these values for BGCT with positive and negative DNA-instability test results showed no statistically meaningful differences. All the BGCT cases negative with DNA-instability test were negative for cmyc expression. Among the BGCT cases positive with DNA-instability test, 18 cases (94.7%) showed cortical bone destruction by computed tomography (CT), and 5 cases (26.3%) showed extra-osseous expansion. No such radiographic changes were detected among the BGCT cases negative with DNA-instability test.
Among 18 BGCT cases with cortical bone destruction, 5 cases (27.8%) showed tumor recurrences, and 2 cases (11.1%) showed lung metastases. These results indicate that the majority of BGCT cases are malignant and the detection of cortical bone destruction by CT is a sensitive clinical marker to detect them.
Key words: Bone giant cell tumor, DNA-instability, Cortical bone defect, Malignancy, Prognosis
Immunohistochemical and Semiquantitative Immunoblot Analyses of Nm23-H1 and H2 Isoforms in Normal Human Tissues
Benio Tsuchiya 1, Yuichi Sato 1, Takeshi Urano 3, Hideo Baba 3, Hiroshi Shiku 4 and Toru Kameya 2
1 Department of Pathology, Kitasato University School of Allied Health Science and 2 Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, 3 Department of Oncology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8102 and 4 Department of Internal Medicine, Faculty of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514-0001
Received for publication June 8, 1998 and in revised form October 30, 1998
The total amount of nm23 protein, the relative ratios of H1 and H2 isoforms (H2/H1) and the localization of these proteins in human normal tissues were studied by a semiquantitative immunoblot technique followed by densitometry and immunohistochemistry with monoclonal antibody against nm23 protein (Pan-242). All tissues contained both isoforms recognized as the 20.5 kD H1 protein and 18.5 kD H2 protein by immunoblotting. Nm23 protein was abundant in liver, kidney and adrenal gland tissue, and scarce in heart and muscle. H2 levels were always higher than H1, but the isoform ratios (H2/H1) were variable from tissue to tissue. Immunostaining revealed that nm23 protein was predominantly present in cytoplasm and the pattern of staining was homogeneous in parenchymal cells of the liver, pancreas and colonic mucosa and heterogeneous in gastric mucosa and kidney. These results demonstrated that the levels of nm23 protein and the H2/H1 ratios and distribution of isoforms were different in each tissue, and suggests that, when the alterations of nm23 gene expression in tumor tissues are examined, the levels and ratios in non-tumorous tissues surrounding the tumor nest should be considered.
1 Department of Obstetrics and Gynecology and 2 2nd Department of Pathology, Yamanashi Medical University, Yamanashi 409-3898 and 3 Institute of Medical Science, University of Tokyo, Tokyo 108-0071
Received for publication July 13, 1998 and in revised form September 18, 1998
To elucidate the physiological roles during pregnancy of vascular endothelial growth factor (VEGF), we examined the temporal and spatial expression of VEGF protein and mRNA in the rat uterine and placental tissues throughout pregnancy. The uterine tissue of nonpregnant rats, pregnant rats, and the rat placental tissue were examined by Northern blotting, in situ hybridization and immunohistochemistry. Northern analysis revealed the existence of VEGF mRNA in the uterus and placenta regardless of the stage of estrous cycle or pregnancy. The results of in situ hybridization and histochemistry for nonpregnant rat specimens showed that VEGF mRNA and protein accumulated in myometrial smooth muscle cells, glandular epithelial cells, stromal cells, and endometrial epithelial cells during each stage of the estrous cycle. The level of accumulation increased in all but the myometrial smooth muscle cells on estrus, and further increased during pregnancy. Furthermore fetal compartments such as trophoblasts, trophoblast giant cells, vitelline epithelial cells, and amnion were labeled more strongly than the maternal compartment.
In conclusion, the spatial expression of VEGF was relatively constant in rat maternal uterus during the estrous cycle and during pregnancy; however, there was a temporal change of VEGF expression.
Key words: Vascular endothelial growth factor, Rat uterus and placenta, In situ hybridization, Immunohistochemistry
Immunocytochemical Localization of Connexin 43 and E-Cadherin in I-407 Cells Mediated by cAMP
Louis Yuge 1, Masao Yamamoto 2, Seiichi Kawamata 1 and Katsuko Kataoka 2
1 Institute of Health Sciences and 2 Department of Anatomy, Hiroshima University School of Medicine, Hiroshima 734-8551
Received for publication July 23, 1998 and in revised form October 12, 1998
The formation of gap junction and the expression of E-cadherin were studied by immunocytochemistry in a cultured intestinal-407 (I-407) cell colonies. These cells scarcely expressed connexin 43 (Cx43) and E-cadherin at a non-stimulated condition. When the cells were stimulated with forskolin and Ro-20-1724, a strong correlation was found between immunocytochemical staining of E-cadherin and that of Cx43 at cell-to-cell contact areas. The length of E-cadherinpositive contact areas increased with time in the stimulated condition. On the other hand, the number of gap junctional plaques in the E-cadherin-expressed areas increased significantly 2 to 8 hr after the stimulation and remained the same level until 24 hr. Both immunostainings were more intense in the central area of the colony than in the peripheral area. Since the administration of forskolin and Ro-20-1724 has been known to elevate cAMP level, these results suggest that (1) the expression of both E-cadherin and gap junctions in cell-to-cell contact is induced by the elevation of cAMP and (2) the effect of cAMP varies by the location of cells in the colony which may reflect the state of cell differentiation.
Key words: Gap junction, Connexin, E-cadherin, Epithelial cells, cAMP, Immunocytochemistry
Department of Industrial Chemistry, Tokai University School of Engineering, Hiratsuka, Kanagawa 257-1207 and *Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193
Received for publication August 24, 1998
In order to investigate the exact mechanism of the antitumor activity of two representative flavonoids, quercetin and genistein, an immunohistochemical study and the supportive biochemical analyses on microtubule assembly and disassembly were performed using hormone refractory human prostate cancer cells in culture (PC3).
Quercetin administration caused distinct morphological changes at a concentration of 20 $\mu$M. Similar morphological changes, cytoplasmic distention and rounding, were observed with vinblastine administration at a lower concentration, but cells treated with genistein were not much different from the control cells. On immunohistochemical observation of $\alpha$-tubulin by a CLSM (Confocal Laser Scanning Microscopy), microtubules were exhibited as fine linear structures running regularly along the long axes of the control and genistein-treated cells. In quercetin-treated cells, however, $\alpha$-tubulin microtubules were distributed in a disorganized manner showing ``criss-cross'' patterns and focal aggregations. This feature was quite similar to that of vinblastinetreated cells. This immunohistochemically demonstrated microtubule disassembly was substantiated by semi quantitation of microtubule disassembly done by western blot analysis of $\alpha$-tubulin which showed a distinct reduction of the polymerized microtubules in the quercetin- and vinblastine-treated cells. Furthermore, in vitro microtubule assembly tests proved that quercetin has definite inhibitory action, though it was with a lower potency than vinblastine, for the microtubule assembly through its direct interaction with tubulin molecules.
Department of 1 Obstetrics and Gynecology and 2 Anatomy, Jichi Medical School, Tochigi, 329-0498
Received for publication September 3, 1998 and in revised form October 15, 1998
The purpose of this study was to characterize the ultrastructural and enzyme-histochemical features of Hofbauer cells in the placental villi of second-trimester abortions. Hofbauer cells in the abortive placenta had more numerous cytoplasmic processes and more prominent phagosomes as compared to gestational age-matched normal control placenta. Acid phosphatase activity was demonstrated in both the lysosomes and on phagosomal membranes of these cells. Our results indicate that Hofbauer cells in second-trimester abortions are activated or stimulated in situ. These activated Hofbauer cells might play important roles in the pathophysiology of preterm parturition.
Antigen Retrieval with Urea for Immunostaining of NOSs in Paraffin Sections of the Rat Brain
Masaru Kimura and Takashi Nakano
Department of Anatomy, Aichi Medical University, Nagakute-cho, Aichi-gun, Aichi 480-1103
Received for publication October 2, 1998
The effects of several antigen retrieval methods on immunostaining of NOSs in paraffin-embedded rat brain tissues were studied in combination with the influence of two fixatives, 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4), and 70% ethanol. The first antibodies for NOSs were purchased from AffinityBioreagents Co., Ltd., New Jersey. Without any antigen-retrieval treatment, paraffin sections of the rat brain fixed with both PFA and ethanol were never immunostained. Only the ethanol fixative was useful for antigen retrieval in immunostaining of NOSs. In antigen retrieval with microwave heating, retrieval solutions such as water (except the one containing 6 M of urea), Tris-HCl buffers at lower pH ($\leq$7.6) with or without urea, and a citrate buffer had no effect. However, a Tris-HCl buffer at pH 9.5 was effective at least for eNOS, and an addition of 5% urea to it enhanced this effect. For both bNOS and eNOS immunostaining, the most effective method was to preincubate sections in an aqueous solution of urea (6 M) for 4 to 12 hr prior to immunostaining. The sections treated with 6 M urea solution showed strong immunostaining reactivity for bNOS in neurons dispersively distributed in the cerebral cortex and in nuclei of the brain stem. With the same pretreatment of sections, eNOS immunoreactivity was also clearly detected on the wall of small blood vessels and capillaries in the parenchyma of the brain.
Key words: Immunohistochemistry, Antigen retrieval, Urea, NOS, Rat brain