bluuebunny/crossref_metadata_2025 huggingface dataset:
Ghrelin Is Expressed in a Novel Endocrine Cell Type in Developing Rat Islets and Inhibits Insulin Secretion from INS-1 (832/13) Cells
Authors: Wierup N. , Yang S. , McEvilly R. J. , Mulder H. , Sundler F.
Published: April 2011

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the β-cell.
Immunofluorescence and Confocal Laser Scanning Microscopy of Chronic Myeloproliferative Disorders on Archival Formaldehyde-fixed Bone Marrow
Authors: Suetterlin Rosmarie , Baschong Werner , Hubert Laeng R.
Published: April 2011

Spatial analysis of the histoarchitecture and photographic documentation at high resolution are the principal advantages of confocal laser scanning microscopy (CLSM) over conventional fluorescence microscopy (CFM) if combined with appropriate software. Restrictions for the use of CFM and CLSM, on the other hand, include nonspecific background fluorescence, fading of photolabile fluorochromes, and both tissue-specific and fixation-induced autofluorescence. Most of those shortcomings can now be avoided. Autofluorescence, the most limiting factor of high-resolution CLSM, was recently controlled also for paraffin sections of archival formaldehyde-fixed tissues. This allowed the present study on cytoskeletal fibers and extracellular matrix proteins in both neoplastic cells of myeloproliferative disorders and in medullary stromal cells using CLSM under proper autofluorescence control. By multiple fluorescence labeling, we found that the intracellular smooth muscle α-actin (SMA) fibers and the two extracellular adhesive matrix proteins tenascin and fibronectin vary in their presence in stromal and/or myeloid cells according to the degree of bone marrow fibrosis in chronic myeloproliferative disorders (CMPDs). CLSM offers further insight in our attempts to understand a complex interplay between the two cellular compartments.
Immunocytochemical Localization of Histatins in Human Salivary Glands
Authors: Ahmad Mansur , Piludu Marco , Oppenheim Frank G. , Helmerhorst Eva J. , Hand Arthur R.
Published: April 2011

Histatins are a family of salivary proteins with bactericidal and fungicidal activities that contribute to the innate defense of the oral cavity. Histatins are present in the serous granules of the parotid and submandibular glands. The important role of histatins in saliva, and the limited information on their cellular and subcellular distribution, prompted us to further define the localization of histatins in the major salivary glands. Immunogold-silver staining of 1-μm sections of plastic-embedded tissue with anti-histatin antibody revealed histatin immunoreactivity in the serous acinar cells of the parotid and submandibular glands, the serous demilune cells of the submandibular and sublingual glands, and in occasional intercalated duct cells. No reactivity was seen in mucous cells or in striated or excretory duct cells. Electron microscopic observations of thin sections labeled with anti-histatin and gold-labeled secondary antibodies revealed immunoreactivity associated with the rough endoplasmic reticulum and Golgi complex and in secretory granules of serous acinar and demilune cells. The granules of parotid acinar cells exhibited relatively uniform labeling of their content, whereas the granules of serous cells in the submandibular and sublingual glands showed variable labeling of the dense and light regions of their content. A few intercalated duct cells adjacent to the acinar cells also exhibited labeled granules. These results suggest that the serous cells of the major glands are the main source of histatins in human saliva. They are also consistent with several previous studies demonstrating the variable distribution of different proteins within the granule content.
Developmental Expression of Pop1/Bves
Authors: Vasavada Trusha K. , DiAngelo Justin R. , Duncan Melinda K.
Published: April 2011

Initial studies have suggested that Pop1/Bves protein is exclusively expressed in the smooth muscle walls of the coronary vessels, implying its possible importance in coronary diseases. However, the mRNA and activity of this gene are detected in both skeletal and cardiac muscles, not coronary smooth muscle, and Pop1/Bves knockout mice have defects in skeletal muscle regeneration. Here we used specific monoclonal antibodies (MAbs) raised against chicken Pop1/Bves and demonstrated the presence of this protein in cardio-myocytes through development and its apparent absence in coronary vessels. Immunostaining of cardiomyocytes cultured in vitro confirmed the membrane localization of this protein in cells that participate in cell adhesion, with significant intracellular staining seen in isolated cells. In skeletal muscle, Pop1 protein becomes detectable at embryonic day (E) 7, coincident with the differentiation of morphologically distinct muscle masses from the limb muscle blastema, but the protein is not found at high levels in the cell membrane of myotubes until E11, coincident with the formation of secondary myotubes from satellite cells. These data support the hypothesis that Pop1/Bves is a cell adhesion molecule present in skeletal and cardiac muscle.
Experimental Evidence for a Direct Cytotoxicity of Loxosceles intermedia (Brown Spider) Venom in Renal Tissue
Authors: Luciano Melissa N. , da Silva Paulo H. , Chaim Olga M. , dos Santos Vera Lucia P. , Franco Célia Regina C. , Soares Maria Fernanda S. , Zanata Silvio M. , Mangili Oldemir C. , Gremski Waldemiro , Veiga Silvio S.
Published: April 2011

Brown spider ( Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.
Expression and Localization of the Transcription Factor JunD in the Duct System of Mouse Submandibular Gland
Authors: Hipkaeo Wiphawi , Wakayama Tomohiko , Yamamoto Miyuki , Iseki Shoichi
Published: April 2011

We studied the expression and localization of JunD, a component of the transcription factor activator protein-1 (AP-1), in the mouse submandibular gland with immunoblotting and immunohistochemistry. In adult mice, all seven Jun and Fos family members constituting the AP-1 complex were expressed more abundantly in the female gland than in the male gland, and JunD was the most abundant of the members. Immunoreactivity for JunD was localized exclusively in the duct system of the gland, in which it was localized to the nuclei of intercalated duct (ID) cells and a subpopulation of striated duct (SD) cells located adjacent to ID. In contrast, granular convoluted tubule (GCT) cells, which are much more abundant in the male gland, were devoid of JunD. During postnatal development of the male gland, JunD was lost from the duct cells as they differentiated to GCT cells at 3-5 weeks postpartum. When GCT differentiation was induced in adult female gland by testosterone administration, many JunD-negative SD cells were temporarily induced to express JunD after 6-24 hr, but those cells lost JunD as they completely converted to GCT cells by 48 hr. These results suggested that JunD is involved in the differentiation of the duct system of mouse submandibular gland, in which there is crosstalk between the androgen/androgen receptor system and the AP-1 complex.
Cell Type-specific Expression of β-Carotene 15,15′-Mono-oxygenase in Human Tissues
Authors: Lindqvist Annika , Andersson Stefan
Published: April 2011

We studied the cell type-specific expression of human β-carotene 15,15′-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.
Expression of the Homeobox Gene Barx2 in the Gut
Authors: Sander Guy R. , Powell Barry C.
Published: April 2011

Barx2 is a member of the Bar class of homeobox genes and has been shown to regulate specific cell adhesion molecules, L1, Ng-CAM, N-CAM, and cadherin 6. By Northern blotting and in situ hybridization, we show that Barx2 is expressed throughout the gut and is located in epithelial cells of the proliferative and differentiative regions of the stomach, esophagus, and intestine. Barx2 was expressed in muscle cells of the muscularis externa and also showed a graded pattern of expression in intestinal enterocytes, decreasing in a crypt-to-villous direction. We speculate that Barx2 may regulate cell adhesion molecules in epithelial cells of the gut.
Immunohistochemical and mRNA Localization of the Angiotensin II Receptor Subtype 2 (AT2) in Follicular Granulosa Cells of the Rat Ovary
Authors: Obermüller Nicholas , Gentili Monika , Gauer Stefan , Gretz Norbert , Weigel Michael , Geiger Helmut , Gassler Nikolaus
Published: April 2011

A local renin-angiotensin system (RAS), including specific angiotensin II receptor subtypes, is present in the rat ovary. Immunohistochemistry using a polyclonal antibody and mRNA in situ hybridization were performed on perfusion-fixed, paraffin-embedded ovaries obtained from untreated sexually mature, normally cycling rats. Immunofluorescent staining revealed an exclusive and distinct labeling of follicular granulosa cells showing a plaque-like expression pattern at the cell borders, being detectable in different stages of atretic degeneration. On adjacent sections the expression of the respective mRNA could be shown in granulosa cells of the same follicle. The AT2 receptor may be implicated in the ovarian atretic process by influencing follicular cell-cell communication.
The Role of Hoxa3 Gene in Parathyroid Gland Organogenesis of the Mouse
Authors: Kameda Yoko , Arai Yuta , Nishimaki Toshiyuki , Chisaka Osamu
Published: April 2011

Mice with a targeted deletion of the Hoxa3 gene have defects of derivatives of the third branchial arch and pouch. To address the role of the Hoxa3 gene in parathyroid organogenesis, we examined the third pharyngeal pouch development by immunohistochemistry (IHC) using the secretory protein (SP)-1/chromogranin A antiserum, which recognizes the parathyroid from its initial formation onward. At embryonic day (E) 11.5, the SP-1/chromogranin A-immunoreactive primary rudiment of the parathyroid appeared in the cranial region of the third pharyngeal pouch of wild-type embryos. In Hoxa3-null mutants, the third pharyngeal pouch was normally formed but failed to differentiate into the parathyroid rudiment, showing no immmunoreactivity for SP-1/chromogranin A. Classic studies using chick-quail chimeras have demonstrated that the ectomesenchymal neural crest cells are required for proper development of the pharyngeal pouch-derived organs, including the thymus and parathyroid glands. To visualize the migration and development of mesenchymal neural crest cells in Hoxa3 mutants, the heterozygotes were crossed with connexin43–lacZ transgenic mice in which β-galactosidase expression was specific to the neural crest cells. In Hoxa3 homozygotes and in wild types, ectomesenchymal neural crest cells densely populated the pharyngeal arches, including the third one, and surrounded the third pouch epithelium. These results indicate that lack of the Hoxa3 gene affects the intrinsic ability of the third pharyngeal pouch to form the parathyroid rudiment and has no detectable effect on the migration of neural crest cells.
Tissue-specific Expression and Subcellular Distribution of Murine Glutathione S-transferase Class Kappa
Authors: Thomson Rachel E. , Bigley Alison L. , Foster John R. , Jowsey Ian R. , Elcombe Clifford R. , Orton Terry C. , Hayes John D.
Published: April 2011

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1–1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.
Developmental Expression of Otoconin-22 in the Bullfrog Endolymphatic Sac and Inner Ear
Authors: Yaoi Yuichi , Onda Tomoaki , Hidaka Yoshie , Yajima Shinya , Suzuki Masakazu , Tanaka Shigeyasu
Published: April 2011

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog ( Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.
Intracellular Localization of Ornithine Decarboxylase and Its Regulatory Protein, Antizyme-1
Authors: Schipper Raymond G. , Cuijpers Vincent M.J.I. , de Groot Linda H.J.M. , Thio Marco , Verhofstad Albert A.J.
Published: April 2011

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocyto-plasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.
Improvement of Combined FISH and Immunofluorescence to Trace the Fate of Somatic Stem Cells after Transplantation
Authors: Donadoni Chiara , Corti Stefania , Locatelli Federica , Papadimitriou Dimitra , Guglieri Michela , Strazzer Sandra , Bossolasco Patrizia , Salani Sabrina , Comi Giacomo P.
Published: April 2011

Fluorescence in situ hybridization (FISH) combined with immunohistochemistry of tissue-specific markers provides a reliable method for characterizing the fate of somatic stem cells in transplantation experiments. Furthermore, the association between FISH and fluorescent gene reporter detection can unravel cell fusion phenomena, which could account for apparent transdifferentiation events. However, despite the widespread use of these techniques, they still require labor-extensive protocol adjustments to achieve correct and satisfactory simultaneous signal detection. In the present paper, we describe an improvement of simultaneous FISH and immunofluorescence detection. We applied this protocol to the identification of transplanted human and mouse hematopoietic stem cells in murine brain and muscle. This technique provides unique opportunities for following the path taken by transplanted cells and their differentiation into mature cell types.
Altered Expression of Aquaporins in Bullous Keratopathy and Fuchs' Dystrophy Corneas
Authors: Kenney M. Cristina , Atilano Shari R. , Zorapapel Nadia , Holguin Bret , Gaster Ronald N. , Ljubimov Alexander V.
Published: April 2011

Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and β2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease.
Expression of Glutamate Transporters in Rat Cardiomyocytes and Their Localization in the T-tubular System
Authors: Kugler Peter
Published: April 2011

Glutamate and aspartate play important roles in the intermediary metabolism of the myocardium and have been shown to improve cardiac recovery after hypoxia or ischemia. Limited data are available about the expression of glutamate transporters that are involved in the uptake of glutamate and aspartate in cardiomyocytes. In this study, non-radioactive in situ hybridization (ISH) using complementary RNA probes was applied to detect the glutamate transporters GLT1 variant (GLT1v) and EAAC1 mRNA in rat cardiomyocytes. The transporter proteins were demonstrated by Western blotting and immunocytochemistry using affinity-purified antibodies against transporter peptides. ISH and immunocytochemistry showed that both glutamate transporters are coexpressed in cardiomyocytes. The ISH labeling indicates the distribution of transporter mRNA throughout the cytoplasm of cardiomyocytes. GLT1v and EAAC1 proteins, which showed in Western blots a molecular mass of ~60 kD, are strongly enriched and colocalized in the transverse (T)-tubular system of cardiomyocytes. These results may indicate that glutamate/aspartate uptake into cardiomyocytes could be mediated by the high-affinity transporters GLT1v and EAAC1. A high efficiency of glutamate/aspartate transport into cardiomyocytes could be achieved by their localization in the T-tubular system, which consists of tubular invaginations of the sarcolemma extending deep into the cell.
The Expression of Mannose Receptors in Skin Fibroblast and Their Involvement in Leishmania (L.) amazonensis Invasion
Authors: Hespanhol Renata C. , Soeiro Maria de Nazaré C. , Meuser Marcos B. , Meirelles Maria de Nazareth S.L. , Côrte-Real Suzana
Published: April 2011

Leishmania are protozoa that invade mononuclear phagocytes with the involvement of different ligand-receptor systems, including mannose receptors. Until now, scant data are available concerning the mechanisms that govern the infection of Leishmania in other host cell types such as fibroblasts. Our aim was to analyze the expression of mannose receptors in primary cultures of skin fibroblasts (SF) further characterizing their role during the invasion of promastigotes of Leishmania (L.) amazonensis. Both fluorescent, light, and electron microscopy assays revealed that SF have mannose receptors since they bound and internalized mannosylated ligands in addition to being positively labeled by fuc-BSA-FITC probes. D-mannose competition assays revealed the participation of mannose receptors during the parasite association with SF presenting upregulated receptor expression during the initial steps of the infection. After longer periods of Leishmania:fibroblasts contact, the modulation noted in the host mannose receptors was reverted concomitantly to the infection control, suggesting that the parasites were required for the alteration maintenance and providing evidences that the SF may display microbicidal mechanisms to control the Leishmania infection.
Localization of Endogenous Furin in Cultured Cell Lines
Authors: Shapiro Jessica , Sciaky Noah , Lee Joseph , Bosshart Herbert , Angeletti Ruth H. , Bonifacino Juan S.
Published: April 2011

Furin is a dibasic endopeptidase responsible for the proteolytic maturation of many precursor proteins in the secretory and endocytic pathways of mammalian cells. The levels of furin expression in most cells are very low, and this has hampered attempts to identify the intracellular compartments in which endogenous furin is localized. We have used a specific antibody reagent to a sequence in the carboxy terminus of furin to perform immunofluorescent staining of mammalian cell lines. This antibody was sensitive enough to detect staining for furin in various cell lines. For the most part, furin staining was confined to a juxtanuclear structure characteristic of the Golgi complex. Analyses by video microscopy and confocal microscopy showed that the distribution of furin was distinct from that of mannosidase II, a marker of the Golgi stack, and most closely resembled that of TGN38, a marker of the trans-Golgi network. Therefore, our results suggest that endogenous furin is predominantly localized to the area of the Golgi complex, most likely within the trans-Golgi network.
DNA Staining for Fluorescence and Laser Confocal Microscopy
Authors: Suzuki Takeshi , Fujikura Keiko , Higashiyama Tetsuya , Takata Kuniaki
Published: April 2011

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).
In Situ Hybridization Analysis of ZPK Gene Expression During Murine Embryogenesis
Authors: Nadeau André , Grondin Gilles , Blouin Richard
Published: April 2011

ZPK is a recently described protein serine/threonine kinase that has been originally identified from a human teratocarcinoma cell line by the polymerase chain reaction and whose function in signal transduction has not yet been elucidated. To investigate the potential role of this protein kinase in developmental processes, we have analyzed the spatial and temporal patterns of expression of the ZPK gene in mouse embryos of different gestational ages. Northern blot analysis revealed a single mRNA species of about 3.5 KB from Day 11 of gestation onwards. In situ hybridization studies demonstrated strong expression of ZPK mRNA in brain and in a variety of embryonic organs that rely on epithelio-mesenchymal interactions for their development, including skin, intestine, pancreas, and kidney. In these tissues, the ZPK mRNA was localized primarily in areas composed of specific types of differentiating cells, and this expression appeared to be upregulated at a time concomitant with the onset of terminal differentiation. Taken together, these observations raise the possibility that the ZPK gene product is involved in the establishment and/or maintenance of a fully cytodifferentiated state in a variety of cell lineages.
Thymic Overexpression of CD40 Ligand Disrupts Normal Thymic Epithelial Organization
Authors: Dunn Robert J. , Luedecker Carrie J. , Haugen Harald S. , Clegg Christopher H. , Farr Andrew G.
Published: April 2011

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.
How to Examine the Antigen-damaging Effect of Sodium Ethoxide on Deplasticized Epoxy Sections
Authors: Brorson Sverre-Henning
Published: April 2011

The purpose of this investigation was to develop a method that could be used to estimate how damaging sodium ethoxide is to different antigens with respect to immunolabeling when epoxy sections are deplasticized. If we obtain weak labeling for an antigen on deplasticized epoxy sections, this might be caused by the damaging effect of the ethoxide solution. It is therefore interesting to develop a method to check if this really is the reason. Fibrin clots and tissues of human kidney and thyroid were embedded in LR White resin. Some thin sections from these specimen blocks were exposed to sodium ethoxide in the same way as epoxy sections are when being deplasticized. Other sections from the same blocks were not exposed to sodium ethoxide. Both categories of sections were immunogold-labeled with anti-fibrinogen, anti-thyroglobulin, anti-IgA, anti-IgG, or anti-IgM. The intensity of immunolabeling of sections treated with ethoxide was compared with the immunolabeling of corresponding sections that were not treated with ethoxide. No significant differences were found in immunolabeling for fibrinogen, IgA, IgG, and IgM. For thyroglobulin, the intensity was approximately 30% less in tissues that were exposed to sodium ethoxide. The practical significance of this method is that we easily can examine the degree to which a given antigen is affected by sodium ethoxide, which is the agent used for deplasticizing epoxy sections.
Quadruple Immunofluorescence: A Direct Visualization Method
Authors: Ferri Gian-Luca , Gaudio Rosa M. , Castello Ida F. , Berger Peter , Giro Gabriele
Published: April 2011

We describe fluorescence immunostaining of four different antigens in the same section. The fluorochrome conjugates used show highest emission in the blue, green, yellow, and red regions of the visible light spectrum, respectively. Specially designed single fluorochrome filter combinations allow selective visualization of each fluorochrome label in turn, without visible crosstalk with the others.
Effects of Shear Stress on Protein Kinase C Distribution in Endothelial Cells
Authors: Hu Ying-Li , Chien Shu
Published: April 2011

We studied the effects of shear stress on protein kinase C (PKC) in cultured human umbilical vein endothelial cells by use of a flow channel and a monoclonal antibody (MAb 1.3) that recognizes the PKC β-isozyme. The fluorescence intensity (FI) of the secondary antibody, crystalline tetramethylrhodamine isothiocyanate, was determined by image analysis. The results on each of five shearing experiments were normalized by using the paired stationary control. After 30-min shearing at 2 N/m2, FI per cell increased to 1.6 times that of control, as did the mean FI per unit cell area. The FI per unit stained area and the stained area/cell area ratio were also increased significantly by shearing. The distribution of immunostaining in each cell was determined for its cortical, cytoplasmic, perinuclear, and nuclear regions. The normalized FI per unit area in all four regions and the stained area/cell area ratio in cortical and cytoplasmic regions were significantly higher in the sheared cells than in control; the increases were greatest in the cortical area. Double staining with rhodamine-phalloidin and MAb 1.3 showed the association of actin with the PKC isozyme in both stationary and sheared cells.
Detection of Human Topoisomerase IIα in Cell Lines and Tissues: Characterization of Five Novel Monoclonal Antibodies
Authors: Kellner Udo , Heidebrecht Hans-Juergen , Rudolph Pierre , Biersack Harald , Buck Friedrich , Dakowski Thomas , Wacker Hans-Heinrich , Domanowski Marcus , Seidel André , Westergaard Ole , Parwaresch Reza
Published: April 2011

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the α-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIβ was ruled out by testing the antibodies on crude extracts from yeast cells expressing the β-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.
Embedding of Bone Samples in Methylmethacrylate: An Improved Method Suitable for Bone Histomorphometry, Histochemistry, and Immunohistochemistry
Authors: Erben Reinhold G.
Published: April 2011

Methylmethacrylate (MMA) embedding of undecalcified bone biopsies is a technique widely used for bone histomorphometry. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Recently, an MMA embedding technique has been reported that preserves enzyme activity and antigenic determinants in bone tissue. We describe here a modification of this embedding method. For our modified MMA embedding process, commercially available methacrylates can be used without purification, and the histologic quality of bone sections is comparable to that of conventionally MMA-embedded bone specimens. The technique reported here can be employed for embedding of larger bone samples and is suitable for histochemical and immunohistological applications as well as for routine bone histomorphometry. By addition of methylbenzoate during infiltration and polymerization of the plastic, the antigenicity of the tissue was improved. As applications of this novel technique, demonstration of alkaline phosphatase and tartrate-resistant acid phosphatase as well as positive labeling of Kupffer cells and osteoclasts with the monoclonal antibody ED1 in sections of liver, tibiae, and vertebrae of 3-month-old rats was demonstrated. The method described here might be useful for the inclusion of histochemical and immunohistological methods into bone histomorphometry.
Fluorescein-labeled Tyramide Strongly Enhances the Detection of Low Bromodeoxyuridine Incorporation Levels
Authors: heusden Jimmy Van , Jong Paul de , Ramaekers Frans , Bruwiere Hélène , Borgers Marcel , Smets Gerda
Published: April 2011

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.
The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH
Authors: Paragas Violette B. , Zhang Yu-Zhong , Haugland Richard P. , Singer Victoria L.
Published: April 2011

We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at ∼360 nm, with emission centered at ∼530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and β-galactosidase mRNA in LacZ+ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor β-chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity.

(J Histochem Cytochem 45:345–357, 1997)
Tissue Distribution of Human gp330/Megalin, a Putative Ca2+-sensing Protein
Authors: Lundgren Stefan , Carling Tobias , Hjälm Göran , Juhlin Claes , Rastad Jonas , Pihlgren Ulla , Rask Lars , Åkerström Göran , Hellman Per
Published: April 2011

We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution.

(J Histochem Cytochem 45:383–392, 1997)
Long-term Oral Application of 5-Bromo-2-Deoxyuridine Does Not Reliably Label Proliferating Immune Cells in the LEW Rat
Authors: Jecker Peter , Beuleke Andrea , Dressendörfer Ingeborg , Pabst Reinhard , Westermann Jürgen
Published: April 2011

To study the lifespan of immune cell populations in the LEW rat, 5-bromo-2-deoxyuridine (BrdU) was administered in the drinking water. After 12 weeks, the epithelium of gut and skin was completely BrdU+. In contrast, thymus, bone marrow, and germinal centers of Peyer's patches contained only a few BrdU+ cells, although most should have been labeled during this time. The lack of labeling was due neither to obvious toxic effects of BrdU on these organs nor to insufficient detection of incorporated BrdU. Analysis of the kinetic pattern of the appearance of BrdU+ cells in bone marrow, blood, spleen, and lymph nodes over 12 weeks revealed that the dosage of BrdU initially was high enough to label the proliferating cells in the bone marrow, but then became too low, although the BrdU uptake of the rats was similar over the entire time. This indicates that in the LEW rat the metabolism of orally applied BrdU changes over time, leading to a reduction in the amount of BrdU available for incorporation into the DNA below a level necessary for labeling all proliferating cells. This effect appears to be species- and strain-dependent, and should be considered when the BrdU technique is used. (J Histochem Cytochem 45:393–401, 1997)
Localization of the Transmembrane 4 Superfamily (TM4SF) Member PETA-3 (CD151) in Normal Human Tissues: Comparison with CD9, CD63, and α5β1 Integrin
Authors: Sincock Paul M. , Mayrhofer Graham , Ashman Leonie K.
Published: April 2011

It has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with β1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, α5β1, and the integrin β1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, α5β1, and β1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules.
Insulin-like Growth Factor I (IGF-I) Induces Unique Effects in the Cytoskeleton of Cultured Rat Glomerular Mesangial Cells
Authors: Berfield Anne K. , Spicer Douglas , Abrass Christine K.
Published: April 2011

Resident glomerular mesangial cells (MCs) have complex cytoskeletal organizations that maintain functional and structural integrity. The ability of cells to replicate, coordinate movement, change shape, and interact with contiguous cells or extracellular matrix depends on cytoskeletal organization. MCs synthesize insulin-like growth factor (IGF-I), express IGF-I receptors, and respond to IGF-I with increased proliferation. We noted that IGF-I treatment of mesangial cells was associated with a change in morphology. Therefore, these studies were undertaken to define specific IGF-I-mediated changes in cytoskeletal protein organization. Rat MCs were propagated from birth in culture without supplemental insulin. Quiescent, subconfluent cultures were treated with IGF-I (100 nM) for 1 hr. Rearrangements in f-actin, α-smooth muscle actin, β-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged, α-smooth muscle actin dissociated from the f-actin bundles and β-actin became polymerized under the leading lamellar edge. Ultrastructural changes were consistent with increased membrane turnover and metabolic activity. The normally sessile mesangial cell was induced by IGF-I to express a wound-healing phenotype characterized by movement and increased pinocytosis. These changes are different from those induced by insulin and have important implications for mesangial cell function.