Gastric Amylin Expression: Cellular Identity and Lack of Requirement for the Homeobox Protein PDX-1. A Study in Normal and PDX-1-Deficient Animals with a Cautionary Note on Antiserum EvaluationTingstedt, Jens-Erik; Edlund, Helena; Madsen, Ole D.; Larsson, Lars-Inge
doi: 10.1177/002215549904700801pmid: 10424881
The gene encoding amylin is implicated in the generation of amyloid in the islets of Langerhans of diabetics and is believed to be regulated by the homeodomain transcription factor PDX-1. Although gastric mucosa also produces amylin, studies on its cellular site of production have yielded highly divergent results, localizing this peptide to either gastrin, serotonin, or somatostatin cells or to combinations thereof. Using region-specific amylin antisera in combination with reverse transcriptase-polymerase chain reaction, we now document that the majority of cells expressing amylin correspond to somatostatin cells. Only a small subpopulation of gastrin cells contained immunoreactive amylin. Studies of PDX-1-deficient mice, which fail to develop gastrin cells while possessing normal numbers of somatostatin cells, revealed no detectable change in gastric amylin expression. These data show that neither normal gastrin cell development nor PDX-1 expression is needed for gastric amylin expression. (J Histochem Cytochem 47:973–980, 1999)
Intracellular Distribution of a Biotin-labeled Ganglioside, GM1, by Immunoelectron Microscopy After Endocytosis in FibroblastsMöbius, Wiebke; Herzog, Volker; Sandhoff, Konrad; Schwarzmann, Günter
doi: 10.1177/002215549904700804pmid: 10424884
A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 μm in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes. (J Histochem Cytochem 47:1005–1014, 1999)
Pretreatment in a High-pressure Microwave Processor for MIB-1 Immunostaining of Cytological Smears and Paraffin Tissue Sections to Visualize the Various Phases of the Mitotic CycleSuurmeijer, Albert J.H.; Boon, Mathilde E.
doi: 10.1177/002215549904700805pmid: 10424885
In many pathology laboratories, both microwave ovens and pressure cookers are used for pretreatment of cytologic smears and paraffin sections to allow MIB-1 staining. For both methods there are two problems. First, the results cannot be used for quantitation because standardization is impossible. Second, the staining results are often suboptimal, resulting in negative staining of cells in the G1- and S-phases. When pretreatment is performed in a microwave processor, allowing microwave heating under pressure, precise temperature monitoring becomes possible. In addition, the importance of the pH of the buffer was studied using a test battery series. Optimal staining is achieved at a temperature of 115C, 10 min, pH 6. This method proved to be highly reproducible. Because the immunostaining results are optimal, the various phases of the cell cycle can be defined in the sections and smears. In addition, the perinucleolar staining of the late G1-phase is optimally visualized and nuclei of the stable pKi-67 pathway can be identified. Under suboptimal conditions, in particular, the number of cells in the late G1-phase are underestimated in the MIB-1 counts. (J Histochem Cytochem 47:1015–1020, 1999)
Quantitative Immunocytochemical Study of Blood-Brain Barrier Glucose Transporter (GLUT-1) in Four Regions of Mouse BrainDobrogowska, Danuta H.; Vorbrodt, Andrzej W.
doi: 10.1177/002215549904700806pmid: 10424886
Application of immunogold cytochemistry revealed polar (asymmetric) distribution of GLUT-1 in mouse brain microvascular endothelia, representing the anatomic site of the blood-brain barrier (BBB). This polarity was manifested by an approximately threefold higher immunolabeling density of the abluminal than the luminal plasma membrane of the endothelial cells. The immunoreaction for GLUT-1 in nonbarrier continuous (skeletal muscle) or fenestrated (brain circumventricular organs) microvascular endothelial cells was absent. In the choroid plexus, the basolateral plasmalemma of the epithelial cells was labeled more intensely than the vascular fenestrated endothelium. Addition of morphometry to the applied immunogold technique makes it possible for even subtle differences to be revealed in the density of immunolabeling for GLUT-1 in blood microvessels located in four brain regions. We found that the density of immunosignals in the microvessels supplying the cerebral cortex, hippocampus, and cerebellum was essentially similar, whereas in the olfactory bulb it was significantly lower. Asymmetric distribution of GLUT-1 in the endothelial plasma membranes presumably leads to a reduced concentration of glucose molecules in the endothelial cells compared to blood plasma and also secures their more rapid transport across the abluminal plasmalemma to the brain parenchyma. (J Histochem Cytochem 47:1021–1029, 1999)
Immunohistochemical Staining Patterns of MUC1, MUC2, MUC4, and MUC5AC Mucins in Hyperplastic Polyps, Serrated Adenomas, and Traditional Adenomas of the ColorectumBiemer-Hüttmann, Anne-Eve; Walsh, Michael D.; McGuckin, Michael A.; Ajioka, Yoichi; Watanabe, Hidenobu; Leggett, Barbara A.; Jass, Jeremy R.
doi: 10.1177/002215549904700808pmid: 10424888
We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyper-plastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyper-plastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum. (J Histochem Cytochem 47:1039–1047, 1999)
Ultrastructural Distribution of 36-kD Microfibril-associated Glycoprotein (MAGP-36) in Human and Bovine TissuesToyoshima, Tetsuhiko; Yamashita, Kayoko; Furuichi, Hiromi; Shishibori, Tsuyoshi; Itano, Toshifumi; Kobayashi, Ryoji
doi: 10.1177/002215549904700809pmid: 10424889
We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis. (J Histochem Cytochem 47:1049–1056, 1999)
Immunocytochemical and In Situ Hybridization Studies of Gastrin After Cisplatin TreatmentWang, Ying; Aggarwal, Surinder K.; Painter, Cory L.
doi: 10.1177/002215549904700810pmid: 10424890
Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in regulating gastric acid production. To study the role of gastrin in this increased gastric acid production after cisplatin treatment, male Wistar rats (100–150 g) were treated with cisplatin (9 mg/kg) in five divided doses over 5 consecutive days. The rats were sacrificed 1, 6, 10, or 15 days after the last treatment. As measured by immunocytochemistry, in situ hybridization, Northern blot, and dot-blot techniques, gastrin was found to be below detectable limits just 1 day after cisplatin treatment. However, 10–15 days after the last injection, the levels for both gastrin and its mRNA gradually recovered to normal. Northern blot studies showed that decreased somatostatin mRNA parallels the changes of gastrin and its mRNA. These results suggest that after cisplatin treatment the increased gastric acid production in rat stomach is independent of gastrin. This decrease of gastrin production is not under the influence of somatostatin, which also decreased after cisplatin treatment. (J Histochem Cytochem 47:1057–1062, 1999)
Ultrastructural Localization of Epithelial Mucin Core Proteins in Colorectal TissuesWinterford, Clay M.; Walsh, Michael D.; Leggett, Barbara A.; Jass, Jeremy R.
doi: 10.1177/002215549904700811pmid: 10424891
Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon. (J Histochem Cytochem 47:1063–1074, 1999)