Journal of Histochemistry & Cytochemistry

Van Driel, Bernard E.M.; Van Noorden, Cornells J.F.

doi: 10.1177/002215549904700501pmid: 10219051

We review here the oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity to detect cancer cells. This inexpensive and rapid assay can be performed within half an hour. Discrimination between cancerous and noncancerous cells is based on a combination of elevated G6PDH activity, decreased super-oxide dismutase (SOD) activity, and decreased lipid peroxidation in cancer cells. The test discriminates between adenomas and carcinomas of the colon with a certainty of >80% and has a high prognostic value for survival of colon cancer patients. Pancreatitis and pancreatic cancer are discriminated with a certainty of 100%. Therefore, the test can be applied by pathologists to provide additional information in difficult cases of diagnosis of cancer and for prognosis.

Palfi, Arpad; Vizi, Sandor; Gulya, Karoly

doi: 10.1177/002215549904700502pmid: 10219052

To investigate the pattern of expression of the three calmodulin (CaM) genes by in situ hybridization, gene-specific [35 S]-cRNA probes complementary to the multiple CaM mRNAs were hybridized in rat brain sections and subsequently detected by quantitative film or high-resolution nuclear emulsion autoradiography. A widespread and differential area-specific distribution of the CaM mRNAs was detected. The expression patterns corresponding to the three CaM genes differed most considerably in the olfactory bulb, the cerebral and cerebellar cortices, the diagonal band, the suprachiasmatic and medial habenular nuclei, and the hippocampus. Moreover, the significantly higher CaM I and CaM III mRNA copy numbers than that of CaM II in the molecular layers of certain brain areas revealed a differential dendritic targeting of these mRNAs. The results indicate a differential pattern of distribution of the multiple CaM mRNAs at two levels of cellular organization in the brain: (a) region-specific expression and (b) specific intracellular targeting. A precise and gene-specific regulation of synthesis and distribution of CaM mRNAs therefore exists under physiological conditions in the rat brain.

Tran, Dien; Stelly, Nicole; Tordjmann, Thierry; Durroux, Thierry; Dufour, Marie Noelle; Forchioni, Arlette; Seyer, René; Claret, Michel; Guillon, Gilles

doi: 10.1177/002215549904700503pmid: 10219053

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8 -PVA. The Gαq/Gα11, PLCβ3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Gαq/Gα11, PLCβ3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Gαq/Gα11 with F-actin.

Valchanov, Kamen P.; Proctor, Gordon B.

doi: 10.1177/002215549904700504pmid: 10219054

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.

Guembe, Laura; Villaro, Ana C.; Treston, Anthony M.

doi: 10.1177/002215549904700505pmid: 10219055

The enzyme PAM is required for activation of many peptide hormones. In adult mouse lung, immunostaining for PAM was located in Clara cells, which constitute most of the epithelial cells of the mouse bronchial/bronchiolar tree. Immunoreactivity appeared for the first time in the epithelium on gestational Day 16, being slight and mostly restricted to the apical cytoplasm. As the lung developed, the labeling became gradually stronger and extended throughout the cell. Smooth muscle of airways and blood vessels, and some parenchymal cells, probably macrophages, also showed PAM immunoreactivity. Of the two enzymatically active domains of PAM, only PHM and not PAL immunoreactivity was found at all stages studied. The early appearance of PAM in developing mouse lung, as well as its presence in a variety of tissues, probably indicates a complex role of this enzyme in pulmonary development and function.

Baba, Takeshi; Ueda, Hideho; Terada, Nobuo; Fujii, Yasuhisa; Ohno, Shinichi

doi: 10.1177/002215549904700506pmid: 10219056

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dynts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The “collared” tubes, which were reported to be localized in nerve terminals in shibirets1 flies and GTPγS-treated synaptosomes, were not observed in the dynts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dynts cells was similar to that in dynwt cells but not to that in dynK44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dynts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dynK44A cells.

Dean, Rachael; Edmondson, Stephanie R.; Burrell, Louise M.; Bach, Leon A.

doi: 10.1177/002215549904700507pmid: 10219057

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (Ml) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after Ml. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to Ml and consequent failure.

Vincent, Andrea M.; Maiese, Kenneth

doi: 10.1177/002215549904700508pmid: 10219058

Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells.

Kataoka, Hiroaki; Suganuma, Tatsuo; Shimomura, Takeshi; Itoh, Hiroshi; Kitamura, Naomi; Nabeshima, Kazuki; Koono, Masashi

doi: 10.1177/002215549904700509pmid: 10219059

We used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration.

Ko, Yu-Shien; Yeh, Hung-I; Rothery, Stephen; Dupont, Emmanuel; Coppen, Steven R.; Severs, Nicholas J.

doi: 10.1177/002215549904700510pmid: 10219060

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.