bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Tumor Necrosis Factor-alpha (TNF-α) in Skeletal Muscle Regeneration
Authors: Collins Rachel A. , Grounds Miranda D.
Published: April 2011

The role of tumor necrosis factor-alpha (TNF-α), an important mediator of the inflammatory response after injury, was investigated in regenerating skeletal muscle. The pattern of expression of TNF-α during muscle regeneration was examined by immunohistochemistry in tissue sections of crush-injured or transplanted muscle autografts and in primary cultures of adult skeletal muscle. TNF-α was highly expressed in injured myofibers, inflammatory cells, endothelial cells, fibroblasts, and mast cells. Myoblasts and myotubes also expressed TNF-α in primary muscle cultures and tissue sections. The essential role of TNF-α and its homologue lymphotoxin-alpha (LT-α) during muscle regeneration was assessed by basic histology in TNF-α(–) and TNF-α(-/-)/LT-α(-/-) mice. No difference was apparent in the onset or pattern of muscle regeneration (i.e., inflammatory response, activation and fusion of myoblasts) between the two strains of null mice or between nulls and normal control mice. However, both strains of null mice appeared more prone to bystander damage of host muscle and regeneration distant from the site of injury/transplantation. Although expression of TNF-α may play an important role in muscle regeneration, the studies in the null mice show that redundancy within the cytokine system (or some other response) can effectively compensate for the absence of TNF-α in vivo. (J Histochem Cytochem 49:989–1001, 2001)
Extensive DNA Fragmentation in Oxyphilic Cell Lesions of the Thyroid
Authors: Volante Marco , Papott Mauro , Gugliotta Patrizia , Migheli Antonio , Bussolati Gianni
Published: April 2011

The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown. (J Histochem Cytochem 49:1003–1011, 2001)
Evidence for Three Fast Myosin Heavy Chain Isoforms in Type II Skeletal Muscle Fibers in the Adult Llama (Lama glama)
Authors: Graziotti Guillermo H. , Ríos Clara M. , Rivero José-Luis L.
Published: April 2011

Skeletal muscle fiber types classified on the basis of their content of different myosin heavy chain (MHC) isoforms were analyzed in samples from hindlimb muscles of adult sedentary llamas ( Lama glama) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry, and quantitative histochemistry of fiber metabolic and size properties. The immunohistochemical technique allowed the separation of four pure (i.e., expressing a unique MHC isoform) muscle fiber types: one slow-twitch (Type I) and three fast-twitch (Type II) phenotypes. The same four major fiber types could be objectively discriminated with two serial sections stained for mATPase after acid (pH 4.5) and alkaline (pH 10.5) preincubations. The three fast-twitch fiber types were tentatively designated as IIA, IIX, and IIB on the basis of the homologies of their immunoreactivities, acid denaturation of their mATPase activity, size, and metabolic properties expressed at the cellular level with the corresponding isoforms of rat and horse muscles. Acid stability of their mATPase activity increased in the rank order IIA>IIX>IIB. The same was true for size and glycolytic capacity, whereas oxidative capacity decreased in the same rank order IIA>IIX>IIB. In addition to these four pure fibers (I, IIA, IIX, and IIB), four other fiber types with hybrid phenotypes containing two (I + IIA, IIAX, and IIXB) or three (IIAXB) MHCs were immunohistochemically delineated. These frequent phenotypes (40% of the semitendinosus muscle fiber composition) had overlapped mATPase staining intensities with their corresponding pure fiber types, so they could not be delineated by mATPase histochemistry. Expression of the three fast adult MHC isoforms was spatially regulated around islets of Type I fibers, with concentric circles of fibers expressing MHC-IIA, then MHC-IIX, and peripherally MHC-IIB. This study demonstrates that three adult fast Type II MHC isoproteins are expressed in skeletal muscle fibers of the llama. The general assumption that the very fast MHC-IIB isoform is expressed only in small mammals can be rejected. (J Histochem Cytochem 49:1033–1044, 2001)
Carbonic Anhydrase II Associated with Plasma Membrane in a Human Pancreatic Duct Cell Line (CAPAN-1)
Authors: Alvarez Laetitia , Fanjul Marjorie , Carter Nicholas , Hollande Etienne
Published: April 2011

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH. (J Histochem Cytochem 49:1045–1053, 2001)
New Molecular Approaches to Tissue Analysis
Authors: Nagle Ray B.
Published: April 2011

The completion of the Human Genome Project will produce new opportunities for analysis of genes and their products in human tissue. The emergence of new technologies will enable investigators to directly examine human tissues for gene deletion, transposition, and amplification. In addition, we will be able to assess the complete gene expression of a tissue by examining the mRNA species using microarray chips. The emerging technologies of laser capture microdissection and RNA amplification enables these procedures to be carried out on groups of a few hundred cells, which will facilitate the examination of heterogeneous lesions. Finally, the application of tissue arrays and the capability of obtaining protein sequences in samples of only a few femtomoles of protein using desorption mass spectroscopy will revolutionize the analysis of protein expression. (J Histochem Cytochem 49:1063–1064, 2001)
Distribution of MAP2 in Hippocampus and Cerebellum of Young and Old Rats by Quantitative Immunohistochemistry
Authors: Di Stefano Giuseppina , Casoli Tiziana , Fattoretti Patrizia , Gracciotti Natascia , Solazzi Moreno , Bertoni–Freddari Carlo
Published: April 2011

The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging. (J Histochem Cytochem 49:1065–1066, 2001)
Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts
Authors: Leitner Karl , Szlauer Roman , Ellinger Isabella , Ellinger Adolf , Zimmer Klaus-Peter , Fuchs Renate
Published: April 2011

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155–1164, 2001)
A Silver Staining Method for Single-cell Gel Assay
Authors: Nadin Silvina B. , Vargas–Roig Laura M. , Ciocca Daniel R.
Published: April 2011

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183–1186, 2001)
Immunohistochemical Analyses of Cell Cycle-related Proteins, Apoptosis, and Proliferation in Pituitary Adenomas
Authors: Nakabayashi Hiromichi , Sunada Ichiro , Hara Mitsuhiro
Published: April 2011

To analyze the cell cycle regulatory mechanisms in the growth of pituitary adenomas, we investigated immunohistochemically the expression of the cell cycle-related proteins cyclin A and p27 in 48 pituitary adenomas. The frequency of apoptosis and the proliferative potential were also examined. The percentage of apoptotic cells was evaluated by immunohistochemical analysis using the anti-single-strand DNA antibody. The proliferative potential was assessed using the anti-Ki-67 antibody. The mean cyclin A labeling index (LI) for the non-recurrent group was 1.03% and for the recurrent group 2.31%. A positive linear correlation between cyclin A LI and Ki-67 LI was found. The mean p27 LI for the non-recurrent group was 67.4% and for the recurrent group 47.0%. There were significant differences in cyclin A LI and p27 LI between the non-recurrent group and the recurrent group. The mean apoptotic rate for the non-recurrent group was 0.87% and for the recurrent group 1.05%. There was no significant difference. Multivariate regression analysis revealed that high cyclin A LI and high Ki-67 LI were significant factors for shorter progression-free survival. The results suggest that the cyclin A LI is a useful prognostic factor in pituitary adenomas. (J Histochem Cytochem 49:1193–1194, 2001)
Cellular Distribution of GAP-43 mRNA in Hippocampus and Cerebellum of Adult Rat Brain by In Situ RT-PCR
Authors: Casoli Tiziana , Di Stefano Giuseppina , Gracciotti Natascia , Giovagnetti Simona , Fattoretti Patrizia , Solazzi Moreno , Bertoni–Freddari Carlo
Published: April 2011

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:1195–1196, 2001)
Gene Expression by Human Articular Chondrocytes Cultured in Alginate Beads
Authors: Chubinskaya Susan , Huch Klaus , Schulze Monika , Otten Lori , Aydelotte Margaret B. , Cole Ada A.
Published: April 2011

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of α1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The α1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.
Localization of the Lipid Receptors CD36 and CLA-1/SR-BI in the Human Gastrointestinal Tract: Towards the Identification of Receptors Mediating the Intestinal Absorption of Dietary Lipids
Authors: Lobo Maria V.T. , Huerta Lydia , Ruiz–Velasco Natividad , Teixeiro Emma , de la Cueva Paloma , Celdrán Angel , Martín–Hidalgo Antonia , Vega Miguel A. , Bragado Rafael
Published: April 2011

The scavenger receptors CLA-1/SR-BI and CD36 interact with native and modified lipoproteins and with some anionic phospholipids. In addition, CD36 binds/transports long-chain free fatty acids. Recent biochemical evidences indicates that the rabbit CLA-1/SR-BI receptor can be detected in enterocytes, and previous studies showed the presence of mRNA for both CLA-1/SR-BI and CD36 in some segments of the intestinal tract. These findings prompted us to study their respective localization and distribution from the human stomach to the colorectal segments, using immunohistochemical methods. Their expression in the colorectal carcinoma-derived cell line Caco-2 was analyzed by Northern blotting. In the human intestinal tract, CLA-1/SR-BI was found in the brush-border membrane of enterocytes from the duodenum to the rectum. However, CD36 was found only in the duodenal and jejunal epithelium, whereas enterocytes from other intestinal segments were not stained. In the duodenum and jejunum, CD36 co-localized with CLA-1/SR-BI in the apical membrane of enterocytes. The gastric epithelium was immunonegative for both glycoproteins. We also found that CLA-1/SR-BI mRNA was expressed in Caco-2 cells and that its expression levels increased concomitantly with their differentiation. In contrast, the CD36 transcript was not found in this colon cell line, in agreement with the absence of this protein in colon epithelium. The specific localization of CLA-1/SR-BI and CD36 along the human gastrointestinal tract and their ability to interact with a large variety of lipids strongly support a physiological role for them in absorption of dietary lipids.
A Comparative Quantitative Analysis of Laminin-5 in the Basement Membrane of Normal, Hyperplastic, and Malignant Oral Mucosa by Confocal Immunofluorescence Imaging
Authors: Haas Michael K. , Berndt Alexander , Stiller Klaus J. , Hyckel Peter , Kosmehl Hartwig
Published: April 2011

Laminin-5 (Ln-5) is a heterotrimeric basement membrane (BM) molecule (α3β3γ2). It is a principal protein constituent of the anchoring filaments, which connect the BM with the hemidesmosomes of the basal keratinocytes and possess a crucial function in keratinocyte adhesion. Confocal immunofluorescence imaging is introduced for a quantitative evaluation of the Ln-5 content in the BM of oral squamous epithelium. The BM of normal oral mucosa was used as a reference (100%) for comparative analysis and showed a nearly uniform Ln-5 immunofluorescence intensity (99–100%). In all hyperplastic lesions of oral mucosa, the Ln-5 immunofluorescence intensity was increased (107–141%). The increased Ln-5 content in the BM of hyperplastic lesions suggests an increased keratinocyte-BM adhesion, possibly resulting in a higher stability of the oral mucosa. In contrast, in the oral squamous cell carcinoma (OSCC) invasive front, the remaining BM segments were characterized by a decrease in Ln-5 immunofluorescence intensity (35–74%). A stronger decrease of Ln-5-linked kerationocyte-BM adhesion correlates with a higher tumor grade. Because in central areas of carcinoma BM segments with a normal Ln-5 content could be demonstrated, the fundamental Ln-5 diminution in BM segments of the invasive front should be considered as an invasion-associated phenomenon.
Immunohistochemical Mapping of Endothelin in the Developing and Adult Mouse Lung
Authors: Guembe Laura , Villaro Ana C.
Published: April 2011

Endothelins (ET) are a family of regulatory peptides displaying, among other abilities, potent constrictor actions. We studied the perinatal time course expression and distribution of ET in the mouse airway epithelium. In fetal mouse, ET-immunoreactivity (IR) appeared earlier (gestational Day 18) in the epithelium of upper (bronchi and large bronchioles) than in lower airways, being scarce and mainly located in the apical cytoplasm. As the lung developed, ET-IR became gradually stronger and extended throughout the cell in both bronchi and bronchioles. ET-IR was found in most airway epithelial cells. Clara cells were positive for ET, whereas ciliated and endocrine cells were not. In adult lungs, part of the myocytes and parenchymal cells also showed ET-IR. In both developing and adult mouse lungs, the cell distribution of ET-IR in the epithelium is compatible with apical and/or basal secretion. The presence of ET in mouse airway epithelium during the perinatal period may indicate a role for ET as a growth factor in lung development and its involvement in control of lung ventilation at birth.
Co-labeling Using In Situ PCR
Authors: Nuovo Gerard J.
Published: April 2011

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight D Nase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1–10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.
Increased Expression of Tenascin-C-binding Epithelial Integrins in Human Bullous Keratopathy Corneas
Authors: Ljubimov Alexander V. , Saghizadeh Mehrnoosh , Pytela Robert , Sheppard Dean , Kenney M. Cristina
Published: April 2011

We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immunofluorescence for TN-C receptors: α2, α8, α9, αVβ3, β1, and β6 integrins, and annexin II. β6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using β2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for α2 and β1 integrins, and for annexin II. α8, α9, and β6 subunits of TN-C receptors, α8β1 α9β1, and αVβ6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. β6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other αVβ6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of β6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially αVβ6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.
Phalloidin-Eosin Followed by Photo-oxidation
Authors: Capani Francisco , Deerinck Thomas J. , Ellisman Mark H. , Bushong Eric , Bobik Marketta , Martone Maryann E.
Published: April 2011

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any perme-abilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.
Different Distribution Patterns of the Two Mannose 6-phosphate Receptors in Rat Liver
Authors: Waguri Satoshi , Kohmura Mari , Kanamori Shiro , Watanabe Tsuyoshi , Ohsawa Yoshiyuki , Koike Masato , Tomiyama Yuji , Wakasugi Masaki , Kominami Eiki , Uchiyama Yasuo
Published: April 2011

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.
Atrial Natriuretic Peptide-like Immunoreactivity in Neurons and Astrocytes of Human Cerebellum and Inferior Olivary Complex
Authors: McKenzie James C. , Juan Yu-Wen , Thomas Charles R. , Berman Nancy E.J. , Klein Robert M.
Published: April 2011

Atrial natriuretic peptide (ANP) has previously been localized in areas of mammalian brain associated with olfaction, cardiovascular function, and fluid/electrolyte homeostasis. Despite the presence of several types of natriuretic peptide receptors in mammalian cerebellum, neither intrinsic nor extrinsic sources of the natriuretic peptides have been described. In this report we describe the immunohistochemical localization of both intrinsic and extrinsic sources for ANP in human cerebellum. ANP-like immunoreactivity (ANP-LIR) was observed in climbing fibers in the cerebellar molecular layer that probably originated from isolated immunopositive neurons of the inferior olivary complex. Intrinsic sources of ANP-LIR included small subpopulations of protoplasmic and fibrous astrocytes and Bergmann glia, as well as Golgi and Lugaro neurons of the granule cell layer. These results suggest that, in addition to its presumptive roles in local vasoregulation, ANP may serve as a modulator of the activity of Purkinje neurons.
Enzyme Cytochemical Techniques for Metabolic Mapping in Living Cells, with Special Reference to Proteolysis
Authors: Boonacker Emil , Van Noorden Cornelis J.F.
Published: April 2011

Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative manner in relation to pathophysiological processes to understand its relevance and the potential impact of its targeting by drugs. Proteases, in particular, are crucial in every aspect of life and death of an organism and are therefore important targets. Enzyme activity in living cells can be studied with various tools. These can be endogenous fluorescent metabolites or synthetic chromogenic or fluorogenic substrates. The use of endogenous metabolites is rather limited and nonspecific because they are involved in many biological processes, but novel chromogenic and fluorogenic substrates have been developed to monitor activity of enzymes, and particularly proteases, in living cells and tissues. This review discusses these substrates and the methods in which they are applied, as well as their advantages and disadvantages for metabolic mapping in living cells.
Tissue Distribution and Membrane Localization of Aquaporin-9 Water Channel
Authors: Nicchia Grazia P. , Frigeri Antonio , Nico Beatrice , Ribatti Domenico , Svelto Maria
Published: April 2011

Aquaporin-9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol, and 5-fluorouracil, a chemotherapeutic agent. With the aim of understanding the pathophysiological role of AQP9, we performed an extensive analysis by Western blotting, RT-PCR, and immunolocalization in rat tissues. Western blotting analysis revealed a major band of approximately 32 kD in testis, liver, and brain. Immunofluorescence showed strong expression of AQP9 in the plasma membrane of testis Leydig cells. In liver, AQP9 expression was found to be sex-linked. Male rats had higher levels of AQP9 than female in terms of both protein and mRNA. Moreover, in female livers the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. No differences in AQP9 expression level related to the age or to protein content of the diet were found, indicating that differences in the liver may be gender-dependent. In the brain, AQP9 expression was found in tanycytes mainly localized in the areas lacking a blood-brain barrier (BBB), such as the circumventricular organs (CVOs) of the third ventricles, the subfornical organ, the hypothalamic regions, and the glial processes of the pineal gland. AQP9 expression in the osmosensitive region of the brain suggests a role in the mechanism of central osmoreception. All these findings show a unique tissue distribution of AQP9 compared to the other known aquaporins.
Altered GalNAc-α-2,6-sialylation Compartments for Mucin-associated Sialyl-Tn Antigen in Colorectal Adenoma and Adenocarcinoma
Authors: Wang Fuan , Goto Masamichi , Kim Young S. , Higashi Michiyo , Imai Kohzoh , Sato Eiichi , Yonezawa Suguru
Published: April 2011

Sialyl-Tn (STn), a mucin-associated disaccharide antigen carried by apomucins such as MUC2, plays an important role in tumor biology. However, little is known about the subcellular localization and compartments involved in STn synthesis. In this study we used immunoelectron microscopy to localize STn and MUC2 apomucin in human colorectal tissues. MUC2 apomucin was localized predominantly in the rough endoplasmic reticulum (RER) in normal colorectal mucosa ( n=6), colorectal adenoma ( n=8), and colorectal adenocarcinoma ( n=10). STn, recognized by monoclonal antibody TKH2, was not readily detectable in normal colorectal mucosa but becomes manifest in both trans-Golgi apparatus and mucin droplets in colorectal adenoma. In colorectal adenocarcinoma, STn was localized not only in late but also in early Golgi compartments, and particularly in some RER lumens. Furthermore, electron microscopic in situ hybridization revealed that gold particles representing MUC2 mRNA are primarily localized over the RER. Our findings indicate that in colorectal adenoma STn sialylation takes place in the trans-Golgi apparatus, whereas in colorectal cancer STn sialylation occurs in all the Golgi compartments and in the RER.
Ultrastructural Staining with Sodium Metaperiodate and Sodium Borohydride
Authors: Lobo Maria V.T. , Alonso F. Javier M. , Arenas Maria I. , Caso Enrique , Fraile Benito , Río Rafael Martín del
Published: April 2011

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed.
Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O
Authors: Möbius Wiebke , Ohno-Iwashita Yoshiko , Donselaar Elly G. van , Oorschot Viola M.J. , Shimada Yukiko , Fujimoto Toyoshi , Heijnen Harry F.G. , Geuze Hans J. , Slot Jan W.
Published: April 2011

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding θ-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-β-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the θ-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.
A Reliable and Convenient Method to Store Ultrathin Thawed Cryosections Prior to Immunolabeling
Authors: Griffith Janice M. , Posthuma George
Published: April 2011

Ultracryotomy of fixed specimens in combination with immunogold labeling is widely used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible. Normally, sections are cut and labeled within 1 day to prevent possible loss or redistribution of soluble antigens within the sections. An increasing demand for more sections and multiple labeling protocols prompted us to investigate the extent to which ultrathin cryosections can be stored. This would render the time spent behind an ultracryomicrotome more efficient and would allow immunogold labeling at a later stage. We investigated whether gelatin plates, 2.3 M sucrose, or 1.0% methyl cellulose/1.2 M sucrose can be used to store thawed frozen sections for a longer period of time. Ultrathin sections of mildly fixed tissue and cultured cells were stored for up to 6 months before immunogold labeling. The preservation of the ultrastructure of stored sections was excellent and was similar to that of immediately processed sections. Importantly, prolonged storage did not affect the labeling intensity.
Detection of Poly(ADP-ribose) Polymerase Activation in Oxidatively Stressed Cells and Tissues Using Biotinylated NAD Substrate
Authors: Bakondi Edina , Bai Péter , Szabó Éva , Hunyadi János , Gergely Pál , Szabó Csaba , Virág László
Published: April 2011

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD+ into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD+), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD+ incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [3H]-NAD incorporation method. The bio-NAD+ assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.
Improved Detection of Substantia Nigra Pathology in Alzheimer's Disease
Authors: Schneider Julie A. , Bienias Julia L. , Gilley David W. , Kvarnberg David E. , Mufson Elliott J. , Bennett David A.
Published: April 2011

The role of substantia nigra pathology in Alzheimer's disease (AD) is uncertain. Detection of pathology may be obscured by intraneuronal neuromelanin and influenced by stains. We determined methods for optimal visualization of nigral pathology in 45 cases of AD. For detection of Lewy bodies (LBs), we compared ubiquitin and α-synuclein immunostains to hematoxylin and eosin (H&E). For neurofibrillary tangles (NFTs) and neuropil threads (NTs), we compared Gallyas silver and paired helical filament (PHF) immunostains, after bleaching of melanin, to modified Bielschowsky, Gallyas, and PHF alone. The number of LB cases was not different using the three stains. However, more LBs per section were detected using α-synuclein ( z=4.88, p<0.001). Twice the number of cases exhibited NFT ( z=8.21; p<0.001) and the mean NFT number per section was 2.8–5.2-fold greater, using Gallyas and PHF after bleaching compared to without bleaching χ2=142.17; p<0.001). More NTs ( z=6.54; p<0.001) were observed with PHF and Gallyas after bleaching. With optimal methods, we found LBs in 27%, NFTs in 89%, and NTs in all 45 AD cases. We show that detection of nigra pathology is influenced by histological method. Clinicopathological studies using these methods are needed to determine the role of nigral pathology in AD.
Developmental and Androgenic Regulation of the Immunocytochemical Distribution of mK1, a True Tissue Kallikrein, in the Granular Convoluted Tubule of the Mouse Submandibular Gland
Authors: Kurabuchi Shingo , Hosoi Kazuo , Gresik Edward W.
Published: April 2011

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immuno-electron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.
Immunohistochemical Detection of Nestin in Pediatric Brain Tumors
Authors: Almqvist Per M. , Mah Richard , Lendahl Urban , Jacobsson Björn , Hendson Glenda
Published: April 2011

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.
Caveolae and Cholesterol Distribution in Vascular Smooth Muscle Cells of Different Phenotypes
Authors: Thyberg Johan
Published: April 2011

Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ER-Golgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipin-sterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or β-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipin-sterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs.
Dietary L-carnitine Stimulates Carnitine Acyltransferases in the Liver of Aged Rats
Authors: Karlic Heidrun , Lohninger Sandra , Koeck Thomas , Lohninger Alfred
Published: April 2011

Aging affects oxidative metabolism in liver and other tissues. Carnitine acyltransferases are key enzymes of this process in mitochondria. As previously shown, the rate of transcription and activity of carnitine palmitoyltransferase CPT1 are also related to carnitine levels. In this study we compared the effect of dietary L-carnitine (100 mg L-carnitine/kg body weight/day over 3 months) on liver enzymes of aged rats (months 21–24) to adult animals (months 6–9) and age-related controls for both groups. The transcription rate of CPT1, CPT2, and carnitine acetyltransferase (CRAT) was determined by quantitative reverse transcription real-time PCR (RTQPCR) and compared to the activity of the CPT1A enzyme. The results showed that the transcription rates of CPT1, CPT2, and CRAT were similar in aged and adult control animals. Carnitine-fed old rats had a significant ( p<0.05) 8–12-fold higher mean transcription rate of CPT1 and CRAT compared to aged controls, adult carnitine-fed animals, and adult controls, whereas the transcription rate of CPT2 was stimulated 2–3-fold in carnitine-fed animals of both age groups. With regard to the enzymatic activity of CPT1 there was a 1.5-fold increase in the old carnitine group compared to all other groups. RNA in situ hybridization also indicated an enhanced expression of CPT1A in hepatocytes from L-carnitine-supplemented animals. These results suggest that L-carnitine stimulates transcription of CPT1, CPT2, and CRAT as well as the enzyme activity of CPT1 in the livers of aged rats.