bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatiotemporal Expression, Distribution, and Processing of POMC and POMC-derived Peptides in Murine Skin
Authors: Mazurkiewicz Joseph E. , Corliss David , Slominski Andrzej
Published: April 2011

In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides β-endorphin, ACTH, β-MSH, and α-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, β-endorphin, β-MSH, and α-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.
Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold-Silver Staining
Authors: Köhler Angela , Lauritzen Bjarne , Noorden Cornelis J.F. Van
Published: April 2011

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold–silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold–silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 μg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold–silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.
Immunohistochemical Localization of Histamine N-Methyltransferase in Guinea Pig Tissues
Authors: Tahara Akihito , Nishibori Masahiro , Ohtsuka Aiji , Sawada Ken , Sakiyama Junko , Saeki Kiyomi
Published: April 2011

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.
A Histomorphometric, Structural, and Immunocytochemical Study of the Effects of Diet-induced Hypocalcemia on Bone in Growing Rats
Authors: Mocetti P. , Ballanti P. , Zalzal S. , Silvestrini G. , Bonucci E. , Nanci A.
Published: April 2011

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal–metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell–matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone–calcified cartilage interface in some cases were thickened but generally did not appear affected at bone–bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption. (J Histochem Cytochem 48:1059–1077, 2000)
Gene Expression Patterns of the Fibroblast Growth Factors and Their Receptors During Myogenesis of Rat Satellite Cells
Authors: Kästner Stefanie , Elias Maria C. , Rivera Anthony J. , Yablonka–Reuveni Zipora
Published: April 2011

Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin+ state. However, FGF6 retards the rapid exit of the cells from the myogenin+ state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1-FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle. (J Histochem Cytochem 48:1079–1096, 2000)
Application of Microwave Technology to the Processing and Immunolabeling of Plastic-embedded and Cryosections
Authors: Rangell Linda K. , Keller Gilbert-André
Published: April 2011

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections. (J Histochem Cytochem 48:1153–1159, 2000)
The Chondrocyte Cytoskeleton in Mature Articular Cartilage: Structure and Distribution of Actin, Tubulin, and Vimentin Filaments
Authors: Langelier Eve , Suetterlin Rosmarie , Hoemann Caroline D. , Aebi Ueli , Buschmann Michael D.
Published: April 2011

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.
Ultrastructural Localization of Myocilin in Human Trabecular Meshwork Cells and Tissues
Authors: Ueda Jun , Wentz–Hunter Kelly K. , Cheng E. Lillian , Fukuchi Takeo , Abe Haruki , Yue Beatrice Y.J.T.
Published: April 2011

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.
Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α
Authors: Enkemann Steven A. , Ward Rita D. , Berger Shelby L.
Published: April 2011

Prothymosin α is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin α appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin α molecules, but not prothymosin α fused to β-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin α shared domains with mobile proteins such as Ran, transportin, and karyopherin β, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin α. The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin α is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.
Expression of the Type I Diabetes-associated Gene LRP5 in Macrophages, Vitamin A System Cells, and the Islets of Langerhans Suggests Multiple Potential Roles in Diabetes
Authors: Figueroa David J. , Hess J. Fred , Ky Bonnie , Brown Sheryl D. , Sandig Volker , Hermanowski–Vosatka Anne , Twells Rebecca C. J. , Todd John A. , Austin Christopher P.
Published: April 2011

LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention.
Expression of Arachidonate 12-Lipoxygenase in Rat Tissues: A Possible Role in Glucagon Secretion
Authors: Kawajiri Hiroo , Zhuang Daming , Qiao Na , Yoshimoto Tanihiro , Yamamoto Miyuki , Iseki Shoichi , Hamaguchi Kazuyuki
Published: April 2011

There are three isoforms of arachidonate 12-lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, α-cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-lipoxygenase in pancreatic α-cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-lipoxygenase cDNA in a glucagon-secreting αTC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets.
The Animal Research Kit (ARK) Can Be Used in a Multistep Double Staining Method for Human Tissue Specimens
Authors: Loos Chris M. van der , Göbel Heike
Published: April 2011

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide–biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.
Distribution of Group II Phospholipase A2 Protein and mRNA in Rat Tissues
Authors: Nyman Kai M. , Ojala Pekka , Laine Veli J.O. , Nevalainen Timo J.
Published: April 2011

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.
Bismuth Autometallography: Protocol, Specificity, and Differentiation
Authors: Danscher Gorm , Stoltenberg Meredin , Kemp Kåre , Pamphlett Roger
Published: April 2011

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique “proton-induced X-ray microanalysis” (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.
Myotube Formation is Delayed but not Prevented in MyoD-deficient Skeletal Muscle: Studies in Regenerating Whole Muscle Grafts of Adult Mice
Authors: White Jason D. , Scaffidi Amelia , Davies Marilyn , McGeachie John , Rudnicki Michael A. , Grounds Miranda D.
Published: April 2011

We compared the time course of myogenic events in vivo in regenerating whole muscle grafts in MyoD(−/−) and control BALB/c adult mice using immunohistochemistry and electron microscopy. Immunohistochemistry with antibodies to desmin and myosin revealed a striking delay by about 3 days in the formation of myotubes in MyoD(−/−) autografts compared with BALB/c mice. However, myotube formation was not prevented, and autografts in both strains appeared similar by 8 days. Electron microscopy confirmed myotube formation in 8- but not 5-day MyoD(−/−) grafts. This pattern was not influenced by cross-transplantation experiments between strains examined at 5 days. Antibodies to proliferating cell nuclear antigen demonstrated an elevated level of replication by MyoD(−/−) myoblasts in autografts, and replication was sustained for about 3 days compared with controls. These data indicate that the delay in the onset of differentiation and hence fusion is related to extended proliferation of the MyoD(−/−) myoblasts. Overall, although muscle regeneration was delayed it was not impaired in MyoD(−/−) mice in this model.
A Highly Sensitive Quantitative Cytosensor Technique for the Identification of Receptor Ligands in Tissue Extracts
Authors: Lenkei Z. , Beaudet A. , Chartrel N. , Mota N. De , Irinopoulou T. , Braun B. , Vaudry H. , Llorens-Cortes C.
Published: April 2011

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1–EGFP receptor in a dose-dependent fashion (EC50 = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1–EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.
Transient Expression of Transglutaminase C During Prenatal Development of Human Muscles
Authors: Lee Suk K. , Chi Je G. , Park Sang C. , Chung Soo I.
Published: April 2011

Tissue transglutaminase (TGase C, TGase II) is known to participate in cellular processes during morphogenesis, differentiation, and development of various prenatal tissues and organs. The expression of TGase C during myoblast proliferation and attachment to external laminae was examined by immunohistochemical (IH) localization at 5–12 weeks of developmental stages of prenatal human muscle in 23 embryos. IH detection using a monospecific antibody to TGase C showed a prominent expression of TGase C in muscle cells as stage- and spatial-specific patterns during an early embryonal period. The myoblasts of intervertebral, tongue, and limb muscles, attached to adjacent cartilaginous skeletons or fibrous fascia, showed a pronounced expression of TGase C at 5–6, 6–7, and 7–8 weeks after fertilization, respectively. The most intense activity of TGase C was observed in some cardiac myoblasts infiltrating into endocardial mesenchyme at 6–7 weeks after fertilization. Although weak staining was detected until 14 weeks after fertilization, the level of TGase C expression in all muscles was significantly decreased after 6–7 weeks, with the exception that the smooth muscle cells of blood vessels and gastrointestinal tract showed diffusely intense staining of TGase C between 5 and 12 weeks after fertilization. Western blotting analysis of the cellular extracts of pooled samples showed a single strong band at 80 kD at 6 weeks after fertilization. This band became weaker after 8–10 weeks of prenatal development. These findings of transient expression of TGase C, which coincides with the development of myoblast anchoring and differentiation, suggest that TGase C plays a role in myoblast attachment to the extracellular laminae during the early embryonal period.
Methods for Imaging Labeled Neurons Together with Neuropil Features in Drosophila
Authors: Tyrer N. Mark , Shepherd David , Williams Darren W.
Published: April 2011

We describe staining protocols for serial semithin sections of Drosophila central ganglia that allow visualization of gene expression in particular neurons with counter-staining to display the ganglion architecture. Green fluorescent protein (GFP), expressed in a subset of sensory neurons from a selected enhancer trap line, is visualized by conventional immunohistochemistry with a peroxidase-linked antibody, and neural architecture is revealed by reduced silver staining. This makes visible in histological sections the same GFP-labeled cells seen with confocal microscopy, but with the especial advantage that neuropil structures are also revealed at the level of individual cells and neuron processes. Not only does this allow the physical relationships among intracellularly labeled neurons to be determined by reference to specific features in the neuropil but it also enables a function to be ascribed provisionally to particular regions of neuropil. These methods have particular utility for mapping morphological information on specific neurons in the context of central nervous system architecture, both in adult Drosophila and during development.
Sensitive and Quantitative Co-detection of Two mRNA Species by Double Radioactive In Situ Hybridization
Authors: Salin Hélène , Maitrejean Serge , Mallet Jacques , Dumas Sylvie
Published: April 2011

A better understanding of biological phenomena involving modulations of gene expression requires quantitative analysis of the expression of several genes in the same structure. For this purpose, we have developed a novel in situ hybridization method to quantify two different mRNA species in the same tissue section simultaneously. Two probes labeled with radioelements of significantly different energies (3H and 33P or 35S) were used to detect the mRNA species. Radioactive images corresponding to the detected mRNA species were acquired with a Micro Imager, a real-time, high-resolution digital autoradiography system. An algorithm was used to process the data such that the initial radioactive image acquired was filtered into two subimages, each representative of the hybridization result specific to one probe. This novel method allows local discrimination and quantification of the respective contributions of each label to each pixel and can therefore be used for quantitative analysis of two mRNAs with a resolution of 15–20 μm.
A New Monoclonal Anti-CD3∊ Antibody Reactive on Paraffin Sections
Authors: Alibaud Laeticia , Llobera Régine , Saati Talal Al , March Michel , Delsol Georges , Rubin Bent
Published: April 2011

We generated a new monoclonal antibody (MAb), F7.2.38, by immunizing mice with CD3∊γδ/CD3ω complexes purified from human T-cells by OKT3 MAb–Sepharose affinity chromatography. Immunoprecipitation experiments and Western blotting analysis showed that MAb F7.2.38 recognized the CD3∊ chain in CD3∊ cDNA-transfected FOX B-cells and in various T-cell lines. Using flow cytometry on permeabilized or intact cells, the epitope was found to be located in the cytoplasmic tail of the CD3∊ chain. Immunohistochemical staining on paraffin-embedded sections showed that the reactivity of MAb F7.2.38 was comparable to that of the commercially available anti-CD3∊ polyclonal antibody. Of the 52 well-characterized T-cell lymphomas, 41 were positive for F7.2.38 (79%), whereas all 37 B-cell lymphomas and 69 non-lymphoid tumors were unreactive. This new anti-CD3∊ antibody would be particularly useful for phenotyping T-cell lymphomas on routinely processed paraffin-embedded tissue sections.
Distribution of H Type 1–4 Chains of the ABO(H) System in Different Cell Types of Human Respiratory Epithelium
Authors: Fujitani Noboru , Liu Yuhua , Okamura Torahiko , Kimura Hiroshi
Published: April 2011

We used three anti-H monoclonal antibodies (MAbs) specific for H Type 1, H Type 2, and H Type 3/4 antigens to investigate the distribution of H Type 1–H Type 4 chains of the ABO(H) histo-blood group in the human respiratory system. Strong staining of H Type 1 chain and weak staining of H Type 2 chain were observed in mucous cells of submucosal glands of bronchial epithelium, which were dependent on the secretor status. No H Type 3/4 chains were detected in mucous cells. Serous cells of submucosal glands of respiratory system showed no staining by three anti-H antibodies. H Type 1 and H Type 3/4 antigens were detected heterogeneously in apical surfaces of bronchial epithelium from secretors but not from nonsecretors. In contrast, basal cells of bronchial epithelium expressed H Type 2 irrespective of the secretor status, probably regulated by the H gene. Some alveolar Type II cells contained only H Types 3/4, which were dependent on the secretor status, whereas alveolar Type I cells had no H antigens. Our results indicated that different cell types in respiratory epithelium expressed different types of carbohydrate chains of histo-blood group antigens under the control of the H or the Se gene.
Developmental Mucin Gene Expression in the Gastroduodenal Tract and Accessory Digestive Glands. I. Stomach: A Relationship to Gastric Carcinoma
Authors: Buisine Marie-Pierre , Devisme Louise , Maunoury Vincent , Deschodt Elisabeth , Gosselin Bernard , Copin Marie-Christine , Aubert Jean-Pierre , Porchet Nicole
Published: April 2011

Studies were undertaken to provide information regarding cell-specific expression of mucin genes in stomach and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of eight mucin genes (MUC1–4, MUC5AC, MUC5B, MUC6, MUC7) in stomach of 13 human embryos and fetuses (8–27 weeks' gestation), comparing these with normal, meta-plastic, and neoplastic adult tissues. These investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. MUC3 mRNA expression can be observed from 10.5 weeks of gestation. MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Normal adult stomach is characterized by strong expression of MUC1, MUC5AC, and MUC6, less prominent MUC2, and sporadic MUC3 and MUC4, without MUC5B and MUC7. Intestinal metaplasia is characterized by an intestinal-type pattern with MUC2 and MUC3 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, abnormal expression of MUC2, and reappearance of MUC5B mRNAs. In conclusion, we have observed that patterns of mucin gene expression in embryonic and fetal stomach could show similarities with some gastric carcinomas in adults. Differences in mucin gene expression in developmental, metaplastic, and neoplastic stomach compared to normal adult stomach suggest a possible regulatory role for their products in gastric epithelial cell proliferation and differentiation.
Secretion of Renin–Angiotensin System (RAS) Components by Normal and Tumoral Lactotropes: A Comparative Study Using Reverse Hemolytic Plaque Assay (RHPA) and Immunoelectron Microscopy
Authors: Vila-Porcile Evelyne , Barret Alain , Corvol Pierre
Published: April 2011

Immunodetection of renin–angiotensin system (RAS) components indicates that there is a local RAS in anterior pituitary cells, particularly in lactotropes. We have attempted to determine if RAS molecules are secreted by lactotropes and the secretory pathways and intracellular sites of maturation. We investigated the secretory activity of individual lactotropes, using the reverse hemolytic plaque assay (RHPA), with GH3B6 tumor cells and normal male rat pituitary cells. We also determined the subcellular distributions of RAS components in these cells. Both tumor and normal cells secreted angiotensinogen, prorenin, renin, angiotensin I, angiotensin-converting enzyme, and angiotensin II, although at different levels. The percentage of secretory cells was generally higher in tumor lactotropes than in normal cells. The subcellular distribution of RAS components obtained by immunoperoxidase was very similar in both cell types, although the intensities of immunoreactivity differed. Cleaved and uncleaved components were found in rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules, all compartments of the secretory pathway. The cleaved components in the RER suggest the existence of early maturation, whereas the presence of uncleaved products in the secretory granules of normal lactotropes might indicate late maturation sites.
Lectin and Proteoglycan Histochemistry of Feline Pacinian Corpuscles
Authors: Sames Klaus , Halata Zdenek , Jojovic Milan , van Damme Els J. M. , Peumans Willy J. , Delpech Bertrand , Asmus Brigitte , Schumacher Udo
Published: April 2011

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were colocalized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.
Expression of the β6 Integrin Subunit Is Associated with Sites of Neutrophil Influx in Lung Epithelium
Authors: Miller Lisa A. , Barnett Nancy L. , Sheppard Dean , Hyde Dallas M.
Published: April 2011

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the β6 integrin, an adhesion molecule associated with proliferation and repair. Expression of β6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the β6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of β6 integrin. In association with C5a instillation and neutrophil influx, β6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of β6 integrin is associated with sites of neutrophil recruitment.
Associations of PKC Isoforms with the Cytoskeleton of B16F10 Melanoma Cells
Authors: Szalay Jeanne , Bruno Patrick , Bhati Ravindra , Adjodha Jennifer , Schueler Deirdre , Summerville Virginia , Vazeos Raina
Published: April 2011

Although PKC plays a major role in regulating the morphology and function of the cytoskeleton, little is known about in situ associations of specific isoforms with the cytoskeleton. We demonstrate that seven PKC isoforms are expressed in B16F10 melanoma cells and show different levels of induction by serum. Using cell cytoskeleton preparations (CSKs), confocal microscopy, and immunocytochemistry, all isoforms show specific patterns of localization to focal contact-like structures (α, δ), very small cytoplasmic granules/vesicles (all isoforms), dense ordered arrays of small granules in the perinuclear region (α, δ), granules/vesicles associated with a homogeneous framework in the cytoplasm adjacent to the nucleus (γ), or irregular-shaped patches of granules at or near the nuclear perimeter (η, θ). In addition, several isoforms are present as cytoplasmic granules/ vesicles in linear or curvilinear arrays (α, δ, ∊, θ). When isoform localization is examined using 3.7% formaldehyde or methanol:acetone, the patterns of localization in CSKs are often difficult or impossible to detect, and many are described here for the first time. Double-labeling experiments with CSK demonstrate that PKC actin co-localizes with punctate α-rich particles above the nucleus, granules of ∊ throughout the cytoplasm, and with θ in irregular-shaped aggregates associated with the nucleus. Vimentin co-localizes with perinuclear granules of δ and β2, and α-tubulin co-localizes with θ in structures at or near the nuclear surface and in microtubules associated with the microtubule organizing center (MTOC). In summary, the present study demonstrates that seven PKC isoforms are endogenously expressed in B16F10 melanoma cells. These isoforms show various levels of induction by serum and specific patterns of association with various components of the detergent-resistant cell cytoskeleton.
Time Course of Osteopontin, Osteocalcin, and Osteonectin Accumulation and Calcification After Acute Vessel Wall Injury
Authors: Gadeau Alain-Pierre , Chaulet Hervé , Daret Danièle , Kockx Mark , Daniel-Lamazière Jean-Marie , Desgranges Claude
Published: April 2011

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.
Differential Subcellular Localization of Zinc in the Rat Retina
Authors: Akagi Takumi , Kaneda Makoto , Ishii Katsuyoshi , Hashikawa Tsutomu
Published: April 2011

In the retina, zinc is believed to be a modulator of synaptic transmission and a constituent of metalloenzymes. To determine whether the intracellular localization of zinc correlates with function, we examined the localization of endogenous zinc in the rat retina using the silver amplification method. By light microscopy, reaction products were detected in the pigment epithelial cells (PE), the inner segment of photoreceptors (IS), the outer nuclear layer (ONL) and the inner nuclear layer (INL), the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and the ganglion cell layer (GC). The heaviest accumulation of precipitate was observed in PE and IS, whereas only a little precipitate was found in GC. When the intracellular zinc was chelated with diethyldithiocarbamate, a small amount of precipitate was observed only in ONL. By electron microscopy, zinc was associated with three compartments. In OPL and IPL, zinc was associated with neural processes, while in PE, IS, INL, and GC it was associated with the Golgi apparatus. In ONL, zinc was associated with the nucleus. Zinc in the neural processes is believed to act as a modulator of synaptic transmission, and zinc associated with the Golgi apparatus is assumed to catalyze metalloenzyme reactions.
Utilizing the Peptidyl-Prolyl Cis-Trans Isomerase Pin1 as a Probe of Its Phosphorylated Target Proteins: Examples of Binding to Nuclear Proteins in a Human Kidney Cell Line and to Tau in Alzheimer's Diseased Brain
Authors: Thorpe Julian R. , Morley Simon J. , Rulten Stuart L.
Published: April 2011

The human parvulin Pin1 is a member of the peptidyl–prolyl cis–trans isomerase group of proteins, which modulate the assembly, folding, activity, and transport of essential cellular proteins. Pin1 is a mitotic regulator interacting with a range of proteins that are phosphorylated before cell division. In addition, an involvement of Pin1 in the tau-related neurodegenerative brain disorders has recently been shown. In this context, Pin1 becomes depleted from the nucleus in Alzheimer's disease (AD) neurons when it is redirected to the large amounts of hyperphosphorylated tau associated with the neurofibrillary tangles. This depletion from the nucleus may ultimately contribute to neuron cell death. Recently we have devised a novel methodology in which exogenous Pin1 is used as a TEM probe for its target proteins. Here we extend this methodology to provide further evidence that Pin1 binds at enhanced levels to mitotic nuclear proteins and to hyperphosphorylated tau in AD brain. We suggest that exogenous Pin1 labeling can be used to elucidate the phosphorylation status of its target proteins in general and could specifically provide important insights into the development of tau-related neurodegenerative brain disorders.
Development and Characterization of 1C6–203, a New Monoclonal Antibody Specific to Human Thymidine Phosphorylase
Authors: Kono Toru , Nishida Miwa , Inagaki Naohito , Tanaka Yutaka , Yoneda Masashi , Kasai Shinichi
Published: April 2011

Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6–203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654–1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6–203 and 654–1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6–203 and 654–1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654–1-stained cancer cells. These results suggest that an epitope recognized by 1C6–203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654–1 is sensitive. Therefore, MAb 1C6–203 might be more suitable than MAb 654–1 for evaluating dThdPase expression in colorectal carcinoma.