Journal of Histochemistry & Cytochemistry

Peters, Barbara; Clausmeyer, Susanne; Obermüller, Nicholas; Woyth, Alexander; Kränzlin, Bettina; Gretz, Norbert; Peters, Jörg

doi: 10.1177/002215549804601101pmid: 9774620

Steroid acute regulatory protein (StAR) plays an essential role in steroidogenesis because it is responsible for the transfer of cholesterol from cellular stores to the inner mitochondrial membrane. We investigated the distribution and regulation of StAR expression in association with aldosterone production in the rat adrenal gland in vivo. Using nonradioactive in situ hybridization, we demonstrate that the outermost five to seven parenchymal cell layers express the StAR gene only weakly and inhomogeneously. The strongest expression is found in the zona fasciculata and zona reticularis. In addition, some cells in the adrenal medulla also stained positively. To differentiate between functionally active glomerulosa and inactive intermediate cells, we compared the expression pattern of StAR with that of aldosterone synthase. The expression of the latter is localized to two or three cell layers only, located immediately below the capsule. However, the cells of the intermedia are capable of expressing both genes prominently, as shown after stimulation with bilateral nephrectomy for 2 days. All zones of the adrenal cortex by then expressed StAR gene to the same extent. This was accompanied by a 50-fold elevated plasma aldosterone concentration. Our data demonstrate that the width of the aldosterone-producing zone can increase within a short period of time by recruiting hormonally inactive cells to steroidogenesis.

Ratcliffe, Elyanne M.; deSa, Derek J.; Dixon, Michael F.; Stead, Ronald H.

doi: 10.1177/002215549804601102pmid: 9774621

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 μg/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.

Krisch, Brigitte; Feindt, Janka; Mentlein, Rolf

doi: 10.1177/002215549804601103pmid: 9774622

We analyzed the internalization of the receptor subtype 2 (sst2) for the neuropeptide somatostatin in glioma cells at the ultrastructural level using an antibody against an extracellular amino acid sequence. Intact cells derived from solid human gliomas or those of the human glioma cell line U343 were receptor-labeled (a) by classical gold immunocytochemistry using a 15-nm gold-labeled second antibody, (b) directly with the sst2 antibody adsorbed to 5-nm colloidal gold, and (c) with the physiological ligand somatostatin conjugated to 5-nm colloidal gold. The receptor was predominantly internalized via uncoated vesicles budding from the cell membrane but only rarely via coated pits, which has been mostly reported for G-protein-coupled, seven transmembrane-domain receptors. In the presence of ligand and sst2 antibody vesicles, tubule-like structures, and multivesicular bodies were labeled in superficial and in perinuclear portions of the cells within the first 30 min. Lysosomal labeling was observed after 30 min and especially after an hour of internalization time. This internalization route is also used to study the directly labeled sst2 antibody or the labeled ligand. However, the late endosomal compartment appears to be reached more rapidly in these latter experiments.

Kurdi-Haidar, Buran; Heath, Dennis; Naredi, Peter; Varki, Nissi; Howell, Steven B.

doi: 10.1177/002215549804601104pmid: 9774623

Human ATPase (hASNA-I) is a novel human gene recently cloned on the basis of homology to the arsA gene of bacteria. Its protein product is an ATPase that is free in the cytoplasm and bound in the perinuclear area and nucleolus in human cells. We prepared the hASNA-I-specific 5G8 monoclonal antibody and used it to investigate the expression of hASNA-I in normal human tissues and breast cancers. hASNA-I was detected immunohistochemically only in the epithelial cells of the liver, kidney, and stomach wall, in the adrenal medulla, in the islet cells of the pancreas, in the red pulp of the spleen, and in cardiac and skeletal muscle. No staining was observed in the uterus, testis, lung, thyroid, cerebellum, and large intestine. Although no staining was also observed in normal breast tissue, all four cases of breast fibroadenomas and all 15 cases of either primary or metastatic breast carcinoma demonstrated increased staining. No embryological or functional common denominator is readily apparent. However, the increased expression in malignant breast cells is of particular interest with respect to the use of this antibody for screening of cytological specimens.

van de Corput, Mariëtte P.C.; Dirks, Roeland W.; van Gijlswijk, Rob P.M.; van Binnendijk, Erica; Hattinger, Claudia M.; de Paus, Roelof A.; Landegent, Jim E.; Raap, Anton K.

doi: 10.1177/002215549804601105pmid: 9774624

Murphy, Susan M.; Pilowsky, Paul M.; Llewellyn-Smith, Ida J.

doi: 10.1177/002215549804601106pmid: 9774625

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for γ-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABAimmunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.

Onoda, Makoto; Inano, Hiroshi

doi: 10.1177/002215549804601107pmid: 9774626

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17β-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/ DMEM plus carboxy-PTIO (an NO scavenger, 100μM) in the presence or absence of LPS (0.5μg/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3μM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 ± 2 kD) and eNOS (152 ± 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.

Davis, R. Eric; Mysore, Veena; Browning, Jared C.; Hsieh, Joseph C.; Lu, Quynh-Anh T.; Katsikis, Peter D.

doi: 10.1177/002215549804601108pmid: 9774627

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an “etheno” (vinyl) bridge. Etheno-NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation.

Laplante, Alain F.; Moulin, Véronique; Auger, François A.; Landry, Jacques; Li, Hui; Morrow, Geneviève; Tanguay, Robert M.; Germain, Lucie

doi: 10.1177/002215549804601109pmid: 9774628

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Mizoguchi, Masayuki; Manabe, Motomu; Kawamura, Yasushi; Kondo, Yukiko; Ishidoh, Kazumi; Kominami, Eiki; Watanabe, Kazutaka; Asaga, Hiroaki; Senshu, Tatsuo; Ogawa, Hideoki

doi: 10.1177/002215549804601110pmid: 9774629