bluuebunny/crossref_metadata_2025 huggingface dataset:
An Optimized Method for In Situ Hybridization with Signal Amplification That Allows the Detection of Rare mRNAs
Authors: Yang Hui , Wanner Ina B. , Roper Stephen D. , Chaudhari Nirupa
Published: April 2011

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61 KB) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues.
Calcium-induced Modification of Protein Conformation Demonstrated by Immunohistochemistry: What Is the Signal?
Authors: Shi Shan-Rong , Cote Richard J. , Hawes Debra , Thu Sandra , Shi Yan , Young Lillian L. , Taylor Clive R.
Published: April 2011

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC.
Bridging the Resolution Gap: Imaging the Same Transcription Factories in Cryosections by Light and Electron Microscopy
Authors: Pombo Ana , Hollinshead Michael , Cook Peter R.
Published: April 2011

The resolution of conventional light microscopy is limited to ∼200 nm in the x- and y-axes and <500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100–200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or “factories,” with diameters of ∼40 nm.
Characterization of Integrin Expression in Islets Isolated from Hamster, Canine, Porcine, and Human Pancreas
Authors: Wang Ren Nian , Paraskevas Steve , Rosenberg Lawrence
Published: April 2011

The reasons for the failure of clinical islet transplantation remain obscure. Islet isolation, however, exposes the islet to variety of cellular stresses, including disruption of the cell-matrix relationship, an event associated with apoptosis. The cell-matrix relationship is characterized by an interaction between cell surface integrin receptors and matrix molecules of the surrounding basement membrane (BM). The purpose of this study was to characterize integrin expression and the distribution of the peri-insular BM in human, porcine, canine, and hamster pancreas, and after routine islet isolation. Whereas islets in the porcine pancreas do not have a demonstrable BM, islets in the human, canine, and hamster pancreas have an almost continuous BM with very little direct exocrine to endocrine cell-cell contact. After islet isolation, the BM was destroyed, only to be reestablished during the period of culture. In the pancreas of all four species, integrin α3 was expressed only on islet cells, and integrin α5 was present on islet cells as well as on acinar, centroacinar, and duct cells. Integrin αV was detected only in human and canine pancreas. Integrin β1 was demonstrated only in the human pancreas. In isolated islets, integrin α3, α5, and αV expression decreased during the culture period and the intensity of the staining was observed to be coincident with the distribution of the BM. In summary, this is the first report of integrin expression in hamster, canine, porcine, and human islets. After islet isolation, the altered islet cell-matrix relationship is reflected both in the decrease in integrin expression and in the destruction of the peri-insular BM. These profound changes will need to be considered as the process of islet isolation for transplantation is refined.
Characterization of a Specific Antibody to the Rat Angiotensin II AT1 Receptor
Authors: Giles Michelle E. , Fernley Ross T. , Nakamura Yoshio , Moeller Ingrid , Aldred G. Peter , Ferraro Tania , Penschow Jennifer D. , McKinley Michael J. , Oldfield Brian J.
Published: April 2011

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.
Cell-specific Expression of Mitochondrial Carbonic Anhydrase in the Human and Rat Gastrointestinal Tract
Authors: Saarnio Juha , Parkkila Seppo , Parkkila Anna-Kaisa , Waheed Abdul , Karttunen Tuomo , Sly William S.
Published: April 2011

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3− to pyruvate carboxylase for the first step in gluconeogenesis and HCO3− to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I.
Differential Expression of Thrombospondin and Cellular Fibronectin During Remodeling in Proliferative Glomerulonephritis
Authors: Barnes Jeffrey L. , Mitchell Ronda J. , Kanalas John J. , Barnes Veronique L.
Published: April 2011

Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, α-smooth muscle actin phenotype, and expression of β-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-β1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, platelet-and macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease.
In Situ Hybridization and Immunohistochemical Analysis of the Expression of Cardiotoxin and Neurotoxin Genes in Naja naja sputatrix
Authors: Lachumanan Ramkumar , Armugam Arunmoziharasi , Durairaj Ponraj , Gopalakrishnakone Ponnampalam , Tan Chee H. , Jeyaseelan Kandiah
Published: April 2011

Secretory processes and their regulation have been extensively studied in mammalian salivary parotid glands. However, little is known regarding the secretory mechanism in the venom glands of snakes, which invariably produce one of the most complex of all animal secretions. The pharmacologically important and toxic components of the Malayan spitting cobra ( Naja naja sputatrix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins (phospholipase A2, PLA2), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as “toxins.” We report here for the first time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period of 8 days after stimulation of venom synthesis by manual “milking” of the venom gland. Immunofluorescence and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes is involved.
Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination Between Nonmalignant and Malignant Cells
Authors: Van Driel Bernard E.M. , Van Noorden Cornells J.F.
Published: April 2011

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.
Differential Distribution and Intracellular Targeting of mRNAs Corresponding to the Three Calmodulin Genes in Rat Brain: A Quantitative In Situ Hybridization Study
Authors: Palfi Arpad , Vizi Sandor , Gulya Karoly
Published: April 2011

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.
Enzyme Histochemistry of Tryptase in Stomach Mucosal Mast Cells of the Mouse
Authors: Valchanov Kamen P. , Proctor Gordon B.
Published: April 2011

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.
Immunocytochemical Study of Endocytotic Structures Accumulated in HeLa Cells Transformed with a Temperature-sensitive Mutant of Dynamin
Authors: Baba Takeshi , Ueda Hideho , Terada Nobuo , Fujii Yasuhisa , Ohno Shinichi
Published: April 2011

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.
Localization of the Insulin-like Growth Factor System in a Rat Model of Heart Failure Induced by Myocardial Infarction
Authors: Dean Rachael , Edmondson Stephanie R. , Burrell Louise M. , Bach Leon A.
Published: April 2011

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.
Direct Temporal Analysis of Apoptosis Induction in Living Adherent Neurons
Authors: Vincent Andrea M. , Maiese Kenneth
Published: April 2011

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.
Distribution of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Human Tissues: Cellular Surface Localization of HAI-1 in Simple Columnar Epithelium and Its Modulated Expression in Injured and Regenerative Tissues
Authors: Kataoka Hiroaki , Suganuma Tatsuo , Shimomura Takeshi , Itoh Hiroshi , Kitamura Naomi , Nabeshima Kazuki , Koono Masashi
Published: April 2011

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.
Connexin Make-up of Endothelial Gap Junctions in the Rat Pulmonary Artery as Revealed by Immunoconfocal Microscopy and Triple-label Immunogold Electron Microscopy
Authors: Ko Yu-Shien , Yeh Hung-I , Rothery Stephen , Dupont Emmanuel , Coppen Steven R. , Severs Nicholas J.
Published: April 2011

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.
Immunocytochemical Localization of Type 5 17 β-Hydroxysteroid Dehydrogenase in Human Reproductive Tissues
Authors: Pelletier Georges , Luu-The Van , Tětu Bernard , Labrie Fernand
Published: April 2011

17β-hydroxysteroid dehydrogenase (17β-HSD) controls the last step in the formation of all androgens and all estrogens. At least six 17β-HSD isoenzymes have been identified. The recently cloned Type 5 17β-HSD transforms 4-dione into testosterone. To gain a better understanding of the role of this enzyme in reproductive tissues, we immunocytochemically localized the enzyme in human male and female reproductive organs. In the ovary of adult premenopausal women (25-40 years of age), immunostaining was found in corpus luteum cells. In the uterus, staining was detected only in the epithelial cells of the endometrium. Immunolabeling was also detected in the mammary gland, a positive reaction being detected in epithelial cells of acini and intralobular ducts as well as in the surrounding stromal cells. In the testis, strong staining was seen in the Leydig cells, and a weak but specific reaction was occasionally detected in Sertoli and germ cells. In the prostate, specific labeling was observed in alveoli and stromal fibroblasts. In alveoli, all the basal cells were generally labeled, whereas the luminal cells exhibited variations in immunoreactivity. In all the reproductive organs examined, specific staining was routinely detected in the walls of blood vessels, including the endothelial cells. These results indicate a cell-specific localization of Type 5 17β-HSD in the different human reproductive organs, thus suggesting new mechanisms of local androgen and estrogen formation that may play an important physiological role.
Quantitative Computer Image Analysis of Chondroitin Sulfate A Expression in Placentas Infected with Plasmodium Falciparum
Authors: Sartelet Hervé , Garraud Olivier , Lorenzato Marianne , Rogier Christophe , Milko-Sartelet Isabelle , Huerre Michel , Gaillard Dominique
Published: April 2011

Most pathological conditions resulting from infection with the human malaria parasite Plasmodium falciparum occur as a consequence of the sequestration by several adhesion molecules of parasite-infected red blood cells (IRBCs). Recent reports have provided evidence that placental vascular endothelial ligands for IRBCs were mostly restricted to chondroitin sulfate A (CSA). The expression of CSA in malaria-infected placentas was investigated in a prospective case-control study in a hypoendemic area (Dakar, Senegal). The tissue distribution of CSA was measured in the terminal villi by immunostaining combined with image processing in 20 infected and 20 noninfected frozen sections of placenta. The villous surface immunostained by anti-CSA antibody was higher in infected than in noninfected placentas ( p<0.03), in placentas with active infection than in those with past chronic infection ( p<0.05), and in infected placentas with positive imprints than in those with negative imprints (not significant; p=0.06). Labeling was found in the extracellular matrix and in endothelial and stromal cells of all the placentas. Syncytiotrophoblast immunostaining was detected in all placentas associated with active or active chronic infection ( n=7) but in only 4/13 placentas with past chronic infection ( p<0.01). The presence of P. falciparum in the imprint was significantly correlated with immunostaining of CSA in syncytiotrophoblasts ( p=0.003). These results suggest that CSA can play an important role in the sequestration of P. falciparum in human placentas during the acute phase of infection.
Tissue and Cell Distribution of the Multidrug Resistance-Associated Protein (MRP) in Mouse Intestine and Kidney
Authors: Peng Kou-Cheng , Cluzeaud Françoise , Bens Marcelle , Duong Van Huyen Jean-Paul , Wioland My Anh , Lacave Roger , Vandewalle Alain
Published: April 2011

The multidrug resistance-associated protein (MRP) that is involved in drug resistance and the export of glutathione-conjugated substrates may not have the same epithelial cell membrane distribution as the P-glycoprotein encoded by the MDR gene. Because intestinal and kidney epithelial cells are polarized cells endowed distinct secreting and absorptive ion and protein transport capacities, we investigated the tissue and cell distribution of MRP in adult mouse small intestine, colon, and kidney by immunohistochemistry. Western blot analyses revealed the 190-kD MRP protein in these tissues. MRP was found in the basolateral membranes of intestinal crypt cells, mainly Paneth cells, but not in differentiated enterocytes. All the cells lining the crypt-villous axis of the colon wall contained MRP. MRP was found in the glomeruli, ascending limb cells, and basolateral membranes of the distal and collecting tubule cells of the kidney but not in proximal tubule cells. Cultured mouse intestinal m-ICc12 cells and renal distal mpkDCT cells that have retained the features typical of intestinal crypt and renal distal epithelial cells, respectively, also possess MRP in their basolateral membranes. The patterns of subcellular and cellular distribution indicate that MRP may have a specific role in the basolateral transport of endogenous compounds in Paneth, renal distal, and collecting tubule cells.
Hakata Antigen, a New Member of the Ficolin/Opsonin p35 Family, Is a Novel Human Lectin Secreted into Bronchus/Alveolus and Bile
Authors: Akaiwa Mina , Yae Yoshiaki , Sugimoto Rie , Suzuki Satoshi O. , Iwaki Toru , Izuhara Kenji , Hamasaki Naotaka
Published: April 2011

Hakata antigen was first reported as a serum protein that reacted with an autoantibody from patients with systemic lupus erythematosus. Recently, it has been found that Hakata antigen is a new member of the ficolin/opsonin p35 family, which is a distinct lectin family, on the basis of homology of structures and the common characteristic of possessing lectin activity. In this study we analyzed the tissue distribution of Hakata antigen. Hakata antigen mRNA and protein were generated in the lung and liver. In the lung, Hakata antigen was produced by both ciliated bronchial epithelial cells and Type II alveolar epithelial cells and was secreted into the bronchus and alveolus. In the liver, Hakata antigen was produced by bile duct epithelial cells and hepatocytes and was also secreted into the bile duct. These results demonstrate that Hakata antigen is a unique lectin protein that exists not only in serum but also in bronchus/alveolus and bile, and indicate that Hakata antigen plays a role in bronchus/alveolus and bile under physiological conditions.
A Quantitative Immunocytochemical Study of Na+, K+-ATPase in Rat Hepatocytes After STZ-induced Diabetes and Dietary Fish Oil Supplementation
Authors: Sennoune Souad , Gerbi Alain , Duran Marie-Josée , Benkoël Liliane , Pierre Sandrine , Lambert Renée , Dodero Frank , Chamlian Albert , Vague Philippe , Maixent Jean-Michel
Published: April 2011

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+, K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+, K+-ATPase catalytic α1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic α1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the α1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+, K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.
HNF-3/Forkhead Homologue-4 (HFH-4) Is Expressed in Ciliated Epithelial Cells in the Developing Mouse Lung
Authors: Tichelaar Jay W. , Wert Susan E. , Costa Robert H. , Kimura Shioko , Whitsett Jeffrey A.
Published: April 2011

HNF-3/forkhead homologue-4 (HFH-4), a transcription factor of the wingedhelix/forkhead family, was detected by immunohistochemistry in tissue of the developing mouse. HFH-4 protein was present in epithelial cells of the lung, trachea, oviduct, and embryonic esophagus, and in ependymal cells lining the spinal column and ventricles of the brain. In lung, trachea, and nose, HFH-4 was expressed in a distinct subset of epithelial cells that also expressed β-tubulin IV, a ciliated cell marker. Cellular sites of HFH-4 and β-tubulin IV expression were distinct from that of Clara cell secretory protein (CCSP), which was detected in nonciliated epithelial cells in the conducting airway of the lung. HFH-4 and β-tubulin IV, but not CCSP, were detected in the respiratory epithelium of thyroid transcription factor-1 (TTF-1) gene-targeted mice. The presence of HFH-4 and β-tubulin IV in TTF-1 gene-targeted mice demonstrates that differentiation of ciliated epithelium does not require TTF-1. Co-localization of HFH-4 and β-tubulin IV staining in various tissues during mouse development supports a role for HFH-4 in the differentiation of ciliated cell lineages.
Microdomain Arrangement of the SERCA-type Ca2+ Pump (Ca2+-ATPase) in Subplasmalemmal Calcium Stores of Paramecium Cells
Authors: Plattner H. , Flötenmeyer M. , Kissmehl R. , Pavlovic N. , Hauser K. , Momayezi M. , Braun N. , Tack J. , Bachmann L.
Published: April 2011

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of ∼8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size (∼106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.
In Vivo Antibody-mediated Modulation of Aminopeptidase A in Mouse Proximal Tubular Epithelial Cells
Authors: Mentzel S. , Dijkman H.B.P.M. , Son J.P.H.F. van , Wetzels J.F.M. , Assmann K.J.M.
Published: April 2011

Aminopeptidase A (APA) is one of the many renal hydrolases. In mouse kidney, APA is predominantly expressed on the brush borders and sparsely on the basolateral membranes of proximal tubular epithelial cells. However, when large amounts of monoclonal antibodies (MAbs) against APA were injected into mice, we observed strong binding of the MAbs to the basolateral membranes, whereas the MAbs bound only transiently to the brush borders of the proximal tubular epithelial cells. In parallel, APA itself disappeared from the brush borders by both endocytosis and shedding, whereas it was increasingly expressed on the basolateral sides. Using ultrastructural immunohistology, we found no evidence for transcellular transport of endocytosed APA to the basolateral side of the proximal tubular epithelial cells. The absence of transcellular transport was confirmed by experiments in which we used a low dose of the MAbs. Such a low dose did not result in binding of the MAbs to the brush borders and had no effect on the presence of APA in the brush borders of the proximal tubular epithelial cells. In these experiments we still could observe binding of the MAbs to the basolateral membranes in parallel with the local appearance of APA. In addition, treatment of mice with chlorpromazine, a calmodulin antagonist that interferes with cytoskeletal function, largely inhibited the MAb-induced modulation of APA. Our studies suggest that injection of MAbs to APA specifically interrupts the normal intracellular traffic of this enzyme in proximal tubular epithelial cells. This intracellular transport is dependent on the action of cytoskeletal proteins.
Connexin45, a Major Connexin of the Rabbit Sinoatrial Node, Is Co-expressed with Connexin43 in a Restricted Zone at the Nodal-Crista Terminalis Border
Authors: Coppen Steven R. , Kodama Itsuo , Boyett Mark R. , Dobrzynski Halina , Takagishi Yoshiko , Honjo Haruo , Yeh Hung-I , Severs Nicholas J.
Published: April 2011

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.
Specificity of the Localization of Transforming Growth Factor-α Immunoreactivity in Colon Mucosa
Authors: Xian Cory J. , Mardell Carolyn E. , Read Leanna C.
Published: April 2011

Transforming growth factor-α (TGF-α) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-α immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-α knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-α) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-α) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-α knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-α molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-α should be used for TGF-α immunohistochemistry in the colon.
Enhanced In Situ Detection of β-Glucuronidase Activity in Murine Tissue
Authors: Kopen Gene C. , Prockop Darwin J. , Phinney Donald G.
Published: April 2011

We outline here a protocol for high-resolution in situ localization of β-glucuronidase in murine tissues processed in glycol methacrylate (GMA). Murine tissues were first stained with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (x-gluc), followed by histological processing in GMA. Retention of the blue indigo reaction product after overnight incubations in x-gluc allowed high-resolution localization of β-glucuronidase activity by brightfield microscopy. When illuminated under darkfield, the x-gluc signal was enhanced, permitting detection even in cells with low-level enzyme activity. This technique offers for the first time a more sensitive enzyme histochemical method of detecting β-glucuronidase activity in animal tissues and also the opportunity to examine expression at high magnification.
RNA Content Differs in Slow and Fast Muscle Fibers: Implications for Interpretation of Changes in Muscle Gene Expression
Authors: Habets Petra E.M.H. , Franco Diego , Ruijter Jan M. , Sargeant Anthony J. , Pereira José A.A. Sant'Ana , Moorman Antoon F.M.
Published: April 2011

Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression. However, total RNA content per gram of a particular fiber type may differ as well. We have tested this possibility in the distinct fiber types of adult rat skeletal muscle. Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content. Quantification of the hybridization showed that the 28S rRNA content decreases in the order i>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type MB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per μg of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis. Consequently, Type I fibers contain five- to sixfold more MHC mRNA per μg of tissue than IIB fibers. These differences are not reflected in the total fiber protein content. This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition. (J Histochem Cytochem 47:995–1004, 1999)
Immunocytochemical Localization of Histamine in Enterochromaffin-like (ECL) Cells in Rat Oxyntic Mucosa: A Transmission Electron Microscopy Study Using Monoclonal Antibodies and Conventional Glutaraldehyde-based Fixation
Authors: Fujiwara Kunio , Bai Gang , Tamura Chihiro , Tsuru Daisuke
Published: April 2011

Histamine (HA), contained in the enterochromaffin-like (ECL) cells of the gastric mucosa in animals, plays an important role in gastric acid secretion, although methods for its exact morphological localization are still lacking. We used a pre-embedding indirect immunoperoxidase approach to define the fine structural localization of HA in rat oxyntic mucosa that was fixed with a glutaraldehyde-based fixative and HA monoclonal antibodies (MAbs AHA-1 and 2). Transmission electron microscopy showed that the peroxidase end-product not only was concentrated in the cores of cytoplasmic granules but also was distributed to a high degree in the cytoplasm peripheral to the granules of the ECL cells. These results suggest that in ECL cells HA is enzymatically synthesized in the cytoplasm, then is transported and stored in the cores of the granules before its release from the basal lamina. The present HA immunoelectron microscopic method with MAbs would be applicable more generally to the ultrastructural identification of HA-containing cells. (J Histochem Cytochem 47:1031–1038, 1999)
Cytochemical Methods for the Detection of Apoptosis
Authors: Willingham Mark C.
Published: April 2011

Detection of apoptotic cell death in cells and tissues has become of paramount importance in many fields of modern biology, including studies of embryonic development, degenerative disease, and cancer biology. In addition to methods that employ biochemical analysis of large populations of cells, cytochemical methods have recently been extensively used both in individual cells and in tissues. Most of these methods exploit properties of dying cells that are more or less specific for the apoptotic process. However, considerable confusion exists over the interpretation of some of these methods and their usefulness in all settings. This review attempts to summarize the more recent advances in cytochemical detection of apoptosis and emphasizes some of the pitfalls that confuse the interpretation of results of these methods.