bluuebunny/crossref_metadata_2025 huggingface dataset:
In Situ Determination of T-cell Receptor Beta Expression Patterns
Authors: Nuovo Gerard J. , Morrison Carl , Porcu Pierluigi , Caligiuri Michael A. , Suster Saul
Published: April 2011

SUMMARY A definitive diagnosis of T-cell lymphoma may be contingent on the rearrangement profile of the T-cell receptor. This is most accurately done by molecular analysis of the beta-chain of the T-cell receptor (TCRβ) by Southern blotting hybridization that requires unfixed tissue. We describe a reverse transcriptase in situ PCR (RT in situ PCR) method that permits the target-specific direct incorporation of the reporter nucleotide into the different transcripts that comprise the TCRβ, using paraffin-embedded, formalin-fixed tissue. Each of the 25 possible Vβ segment rearrangments was documented in three lymph nodes with nonspecific lymphadenitis, with clonal expansion evident in a case of metastatic melanoma. Monoclonal expression was documented in seven tissues diagnostic of a T-cell lymphoma. We analyzed five additional tissues for which a definitive diagnosis of T-cell vs B-cell lymphoma could not be rendered on the basis of histological, immunohistological, and flow cytometric analysis. RT in situ PCR for TCRβ expression with CD3 co-labeling demonstrated which of these lesions was a B-cell-rich T-cell lymphoma. We conclude that the RT in situ PCR methodology will allow the routine determination of monoclonal vs multiclonal expression patterns of the TCRβ using archival paraffin-embedded tissues. (J Histochem Cytochem 49:139–145, 2001)
Spectral Imaging of MC540 During Murine and Human Colon Carcinoma Cell Differentiation
Authors: Siboni Galit , Rothmann Chana , Ehrenberg Benjamin , Malik Zvi
Published: April 2011

SUMMARY We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G1-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147–153, 2001)
Localized Infusion of Tunicamycin in Rat Hemimandibles: Alteration of the Basal Lamina Associated with Maturation Stage Ameloblasts
Authors: Orsini Giovanna , Zalzal Sylvia , Nanci Antonio
Published: April 2011

SUMMARY At the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N-glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectin–gold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N-linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions. (J Histochem Cytochem 49:165–176, 2001)
Fluorescence Properties and Staining Behavior of Monodansylpentane, a Structural Homologue of the Lysosomotropic Agent Monodansylcadaverine
Authors: Niemann Axel , Baltes Jennifer , Elsässer Hans-Peter
Published: April 2011

SUMMARY We have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion-trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells. (J Histochem Cytochem 49:177–185, 2001)
Subcellular Location and Expression Pattern of Autoimmune Regulator (Aire), the Mouse Orthologue for Human Gene Defective in Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy (APECED)
Authors: Halonen Maria , Pelto–Huikko Markku , Eskelin Petra , Peltonen Leena , Ulmanen Ismo , Kolmer Meelis
Published: April 2011

SUMMARY Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), also known as autoimmune polyglandular syndrome Type I (APS1), is an autosomal recessive autoimmune disease caused by mutations in a gene designated as AIRE (autoimmune regulator). Here we have studied the expression of Aire in transfected cell lines and in adult mouse tissues. Our results show that Aire has a dual subcellular location and that it is expressed in multiple immunologically relevant tissues such as the thymus, spleen, lymph nodes, and bone marrow. In addition, Aire expression was detected in various other tissues such as kidney, testis, adrenal glands, liver, and ovary. These findings suggest that APECED protein might also have a function(s) outside the immune system. (J Histochem Cytochem 49:197–208, 2001)
Identification and Isolation of Rat Bone Marrow-derived Mast Cells Using the Mast Cell-specific Monoclonal Antibody AA4
Authors: Jamur Maria Célia , Grodzki Ana Cristina G. , Moreno Andrea N. , de Mello Luciana de Fátima C. , Pastor Maria Veronica D. , Berenstein Elsa H. , Siraganian Reuben P. , Oliver Constance
Published: April 2011

SUMMARY Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the α-subunit of Fc∊RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells. (J Histochem Cytochem 49:219–228, 2001)
A Simple Assay and Histochemical Localization of Transglutaminase Activity Using a Derivative of Green Fluorescent Protein as Substrate
Authors: Furutani Yutaka , Kato Akira , Notoya Michitaka , Ghoneim Magdy A. , Hirose Shigehisa
Published: April 2011

SUMMARY Histidine-tagged green fluorescent protein (His6-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His6-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His6-Xpress). His6-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247–258, 2001)
Identification of the Full-length AE2 (AE2a) Isoform as the Golgi-associated Anion Exchanger in Fibroblasts
Authors: Holappa Katja , Suokas Marko , Soininen Paula , Kellokumpu Sakari
Published: April 2011

SUMMARY Na+-independent Cl−/HCO3− exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid–base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259–269, 2001)
Ultrastructural Localization of Vesicle-associated Membrane Protein(s) to Specialized Membrane Structures in Human Pericytes, Vascular Smooth Muscle Cells, Endothelial Cells, Neutrophils, and Eosinophils
Authors: Feng Dian , Flaumenhaft Robert , Bandeira–Melo Christianne , Weller Peter F. , Dvorak Ann M.
Published: April 2011

Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculo–vacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells.
Occludin and Claudin-1 Concentrate in the Midbody of Immortalized Mouse Hepatocytes During Cell Division
Authors: Kojima Takashi , Kokai Yasuo , Chiba Hideki , Osanai Makoto , Kuwahara Kazuhide , Mori Michio , Mochizuki Yohichi , Sawada Norimasa
Published: April 2011

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.
Synaptotagmin I Expression in Mast Cells of Normal Human Tissues, Systemic Mast Cell Disease, and a Human Mast Cell Leukemia Cell Line
Authors: Kimura Noriko , Shiraishi Shin-ichiro , Mizunashi Kazutoshi , Ohtsu Hiroshi , Kimura Itaru
Published: April 2011

Synaptotagmin I (STG I) is a Ca2+ sensor and one of the synaptic vesicle proteins that mediate exocytosis. To determine the mechanism of release of large granules from mast cells, we studied by immunohistochemistry the presence of STG I in mast cells in normal human tissues simultaneously with the mast cell markers mast cell tryptase (tryptase) and c-kit. The tumor cells of systemic mast cell disease (SMCD) and a human mast cell leukemia cell line (HMC-1) were also examined. Human mast cells in normal tissues and the tumor cells of SMCD expressed STG I as well as mast cell tryptase (tryptase) and c-kit. STG I mRNA and its products in HMC-1 were examined by RT-PCR analysis and immunocytochemistry, respectively. STG I expression in HMC-1 cells was compared with that in cells stimulated and non-stimulated by phorbol 12-myristate 13-acetate and also with that in NB-1 and PC12 cells, known to express STG I. STG I mRNA was detected in both non-stimulated and stimulated HMC-1 cells and in NB-1 and PC12 cells. STG I immunoreactivity was weaker than NB-1 or PC12 immunoreactivity. However, it increased in the stimulated HMC-1 cells. Mast cells expressed STG I in various states. STG I may mediate exocytosis of large granules in mast cells.
The Vesiculo–Vacuolar Organelle (VVO): A New Endothelial Cell Permeability Organelle
Authors: Dvorak Ann M. , Feng Dian
Published: April 2011

A newly defined endothelial cell permeability structure, termed the Vesiculo–Vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.
Immunohistochemical Distribution of CD9, Heparin Binding Epidermal Growth Factor-like Growth Factor, and Integrin α3β1 in Normal Human Tissues
Authors: Nakamura Yasuhiro , Handa Kazuyo , Iwamoto Ryo , Tsukamoto Takahisa , Takahasi Mitsuhiko , Mekada Eisuke
Published: April 2011

The tetra-membrane-spanning protein CD9 forms a complex with a membrane-anchored heparin binding epidermal growth factor-like growth factor (HB-EGF) and integrin α3β1 in some human and monkey cell lines. We show here the immunohistochemical distribution of CD9, HB-EGF, and integrin α3β1 in normal human tissues. Distribution of CD9, HB-EGF, and integrin α3β1 was similar in various tissues, including transitional epithelium, squamous epithelium, thyroid follicular epithelium, adrenal cortex, testis, smooth muscle, and stromal fibrous tissue. However, distribution of the three proteins did not coincide in some tissues, such as lung, liver, kidney, gastric and intestinal epithelium, pancreas, salivary gland, and ovary. In striated muscle, including cardiac muscle, CD9 was present not in the muscle cells themselves but in the endomysium and perimysium, whereas HB-EGF was distributed in the muscle cells themselves. CD9 was distributed in the myelin, but HB-EGF was found in the axon of the peripheral and central nervous systems. Coincident distribution of integrin α3β1 with others was not observed in muscles and neural tissues. In conclusion, there is a possibility of complex formation and functional cooperation of CD9 with HB-EGF and/or integrin α3β1 in several tissues.
Selective Processing of Chromogranin A in the Different Islet Cells in Human Pancreas
Authors: Portela–Gomes Guida Maria , Stridsberg Mats
Published: April 2011

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the four major neuroendocrine cell types of the human pancreas by using double immunofluorescence techniques. The antibodies raised to the N-terminal and midportions of CgA showed, on the whole, stronger immunoreactivity than did the C-terminal antibodies, with a few exceptions. Often the immunoreactivity was stronger in glucagon cells. Insulin cells expressed immunoreactivity to all region-specific antibodies, but glucagon cells were nonreactive to two antibodies. Somatostatin cells reacted only with the C-terminal antibodies (amino acid sequences CgA 411–424), while PP cells were stained with four CgA region-specific antibodies between amino acid sequences 63–195. The cause of these differences may be that the CgA molecule is cleaved, partly masked, or partly translated from CgA mRNA. Microwave treatment improved only the staining with the CgA 361–372 antibodies, which indicates that masking is not the sole or entire cause. Our findings may indicate that the CgA molecule is cleaved in different ways in the various pancreatic endocrine cell types, giving rise to a variety of biologically functional fragments.
Differential Expression of a Stress-modulating Gene, BRE, in the Adrenal Gland, in Adrenal Neoplasia, and in Abnormal Adrenal Tissues
Authors: Miao Ji , Panesar Nirmal S. , Chan Kin-Tak , Lai Fernand M.M. , Xia Ning-shao , Wang Ying-bin , Johnson Philip J. , Chan John Y.H.
Published: April 2011

Genes that modulate the action of hormones and cytokines play a critical role in stress response, survival, and in growth and differentiation of cells. Many of these biological response modifiers are responsible for various pathological conditions, including inflammation, infection, cachexia, aging, genetic disorders, and cancer. We have previously identified a new gene, BRE, that is responsive to DNA damage and retinoic acid. Using multiple-tissue dot-blotting and Northern blotting, BRE was recently found to be strongly expressed in adrenal cortex and medulla, in testis, and in pancreas, whereas low expression was found in the thyroid, thymus, small intestine and stomach. In situ hybridization and immunohistochemical staining indicated that BRE was strongly expressed in the zona glomerulosa of the adrenal cortex, which synthesizes and secretes the mineralocorticoid hormones. It is also highly expressed in the glial and neuronal cells of the brain and in the round spermatids, Sertoli cells, and Ledig cells of the testis, all of which are associated with steroid hormones and/or TNF synthesis. However, BRE expression was downregulated in human adrenal adenoma and pheochromocytoma, whereas its expression was enhanced in abnormal adrenal tissues of rats chronically treated with nitrate or nitrite. These data, taken together, indicate that the expression of BRE is apparently associated with steroids and/or TNF production and the regulation of endocrine functions. BRE may play an important role in the endocrine and immune system, such as the cytokine–endocrine interaction of the adrenal gland.
Differential Distribution of Sialic Acid in α2,3 and α2,6 Linkages in the Apical Membrane of Cultured Epithelial Cells and Tissues
Authors: Ulloa Fausto , Real Francisco X.
Published: April 2011

We used lectin cytochemistry and confocal microscopy to examine the distribution of sialic acid in epithelial cells. Maackia amurensis lectin and Sambuccus nigra agglutinin were used to detect α2,3 and α2,6 sialic acid, respectively. In Caco-2, HT-29 5M12, and MCF-7 cells, which express sialic acid mainly in one type of linkage, the majority of the signal was observed in the apical membrane. In cells that bound both lectins, α2,3 sialic acid was distributed apically, whereas α2,6 sialic acid showed a broader distribution. In IMIM-PC-1 cultures, α2,3 sialic acid was detected mainly in the apical membrane, whereas α2,6 sialic acid was more abundant in the basoleral domain of polarized cells. In these cells, treatment with GalNAc- O-benzyl led to reduced α2,3 levels and to an increase and redistribution of α2,6 to the apical domain. Similarly, sialic acid was predominantly expressed apically in all epithelial tissues examined. In conclusion, (a) sialic acid is mainly distributed to the apical membrane of epithelial cells, (b) there is a hierarchy in the distribution of sialic acids in polarized epithelial cells, i.e., α2, 3 is preferred to α2, 6 in the apical membrane, and (c) IMIM-PC-1 cells are a good model in which to study the regulation of the levels and distribution of sialic acids.
Identification and Purification of Functional Human β-cells by a New Specific Zinc-fluorescent Probe
Authors: Lukowiak Bruno , Vandewalle Brigitte , Riachy Rita , Kerr–Conte Julie , Gmyr Valery , Belaich Sandrine , Lefebvre Jean , Pattou François
Published: April 2011

Pancreatic β-cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable β-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number ( r = 0.98) and between zinc fluorescence and insulin content ( r = 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of β-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for β-cell experimentation, both clinically and for basic research.
δL- and γ-Sarcoglycan Localization in the Sarcoplasmic Reticulum of Skeletal Muscle
Authors: Ueda Hideho , Ueda Kei , Baba Takeshi , Ohno Shinichi
Published: April 2011

Sarcoglycans are transmembrane proteins that are members of the dystrophin complex. Sarcoglycans cluster together to form a complex, which is localized in the cell membrane of skeletal, cardiac, and smooth muscle fibers. However, it is still unclear whether or not sarcoglycans are restricted to the sarcolemma. To address this issue, we examined α-, β-, δ-, and γ-sarcoglycan expression in femoral skeletal muscle from control and dystrophin-deficient mice and rats using confocal microscopy and immunoelectron microscopy. Confocal microscopy of the tissues in cross-section showed that all sarcoglycans were detected under the sarcolemma in rats and control mice. δ- and γ-sarcoglycan labeling demonstrated striations in the longitudinal section, suggesting that the proteins were expressed in the sarcoplasmic reticulum (SR) or transverse tubules (T-tubules). Moreover, such striations of both sarcoglycans were recognized in the dystrophin-deficient mouse skeletal muscle. Double labeling with phalloidin or α-actinin and δ- or γ-sarcoglycan showed different labeling patterns, indicating that δ-sarcoglycan localization was distinct from that of γ-sarcoglycan. Immunoelectron microscopy clarified that δ-sarcoglycan was localized in the terminal cisternae of the SR, while γ-sarcoglycan was found in the terminal cisternae and longitudinal SR over I-bands but not over A-bands. These data demonstrate that δ- and γ-sarcoglycans are components of the SR in skeletal muscle, suggesting that both sarcoglycans function independent of the dystrophin complex in the SR.
The Emergence of Electron Tomography as an Important Tool for Investigating Cellular Ultrastructure
Authors: McEwen Bruce F. , Marko Michael
Published: April 2011

Electron tomography has emerged as the leading method for the study of three-dimensional (3D) ultrastructure in the 5–20-nm resolution range. It is ideally suited for studying cell organelles, subcellular assemblies and, in some cases, whole cells. Tomography occupies a place in 3D biological electron microscopy between the work now being done at near-atomic resolution on isolated macromolecules or 2D protein arrays and traditional serial-section reconstructions of whole cells and tissue specimens. Tomography complements serial-section reconstruction by providing higher resolution in the depth dimension, whereas serial-section reconstruction is better able to trace continuity over long distances throughout the depth of a cell. The two techniques can be combined with good results for favorable specimens. Tomography also complements 3D macromolecular studies by offering sufficient resolution to locate the macromolecular complexes in their cellular context. The technology has matured to the point at which application of electron tomography to specimens in plastic sections is routine, and new developments to overcome limitations due to beam exposure and specimen geometry promise to further improve its capabilities. In this review we give a brief description of the methodology and a summary of the new insights gained in a few representative applications. (J Histochem Cytochem 49:553–563, 2001)
The Cortactin-binding Postsynaptic Density Protein ProSAP1 in Non-neuronal Cells
Authors: Redecker Peter , Gundelfinger Eckart D. , Boeckers Tobias M.
Published: April 2011

Proline-rich synapse-associated protein-1 (ProSAP1) is a neuronal PDZ domain-containing protein that has recently been identified as an essential element of the postsynaptic density. Via its interaction with the actin-binding protein cortactin and its integrative function in the organization of neurotransmitter receptors, ProSAP1 is believed to be involved in the linkage of the postsynaptic signaling machinery to the actin-based cytoskeleton, and may play a role in the cytoskeletal rearrangements that underlie synaptic plasticity. As a result of our ongoing studies on the distribution and function of this novel PDZ domain protein, we now report that the expression of ProSAP1 is restricted neither to neurons and interneuronal junctions nor to the nervous system. Using immunohistochemical techniques in conjunction with specific antibodies, we found that, in the CNS, ProSAP1 can be detected in certain glial cells, such as ependymal cells, tanycytes, subpial/radial astrocytes, and in the choroid plexus epithelium. Moreover, our immunohistochemical analyses revealed the presence of ProSAP1 in endocrine cells of the adenohypophysis and of the pancreas, as well as in non-neuronal cell types of other organs. In the pancreas, ProSAP1 immunoreactivity was also localized in the duct system of the exocrine parenchyma. Our findings demonstrate that, in addition to neurons, ProSAP1 is present in various non-neuronal cells, in which it may play a crucial role in the dynamics of the actin-based cytoskeleton.

(J Histochem Cytochem 49:639–648, 2001)
Changes of AT2 Receptor Levels in the Rat Adrenal Cortex and Medulla Induced by Bilateral Nephrectomy and Its Modulation by Circulating ANG II
Authors: Peters Barbara , Clausmeyer Susanne , Teubner Philipp , Obermüller Nicholas , Kränzlin Bettina , Gretz Norbert , Inagami Tadashi , Peters Jörg
Published: April 2011

We studied regulation of the AT2 receptor by investigating the effect of bilateral nephrectomy (bNX) in Sprague–Dawley rats. The expression of aldosterone synthase (CYP11B2) and AT2 receptor mRNA was detected by nonradioactive in situ hybridization. AT2 receptor mRNA was detected in cells of the first two or three subcapsular cell layers of the zona glomerulosa (ZG) and in the medulla of sham-operated animals. After bNX, the number and area of distribution of AT2 receptor-positive cells increased in the ZG. This was associated with an enlargement of the steroidogenic active ZG and with reduced proliferation rate (sham 5.9 ± 0.9%; bNX 2.4 ± 0.2%; p > 0.02). Infusion of angiotensin II (ANG II; 200 ng/kg/min SC for 56 hr) to bNX rats did not reverse the effect of nephrectomy on the distribution of AT2 receptor expression, although mRNA levels per cell were reduced compared to NX alone. ANG II infusion decreased proliferation rate further (0.4 ± 0.07%; p > 0.001). In the adrenal medulla after bNX, decreased expression of the AT2 receptor was associated with increased proliferation (2.6 ± 0.2% vs 6.6 ± 0.5%). These results demonstrate differential regulation of the AT2 receptor in the adrenal gland and suggest that expression of the AT2 receptor is involved in regulating proliferation and differentiation in the ZG and medulla.

(J Histochem Cytochem 49:649–656, 2001)
Secretion of Carbonic Anhydrase Isoenzyme VI (CA VI) from Human and Rat Lingual Serous von Ebner's Glands
Authors: Leinonen Jukka , Parkkila Seppo , Kaunisto Kari , Koivunen Petri , Rajaniemi Hannu
Published: April 2011

Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis.

(J Histochem Cytochem 49:657–662, 2001)
The Use of Counterflow Centrifugation to Enrich Gonadotropes and Somatotropes
Authors: Childs Gwen V. , Unabia Geda
Published: April 2011

Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.

(J Histochem Cytochem 49:663–664, 2001)
Cyclin T2A Gene Maps on Human Chromosome 2q21
Authors: De Luca Antonio , Tosolini Alessandra , Russo Patrizia , Severino Anna , Baldi Alfonso , De Luca Luca , Cavallotti Ivan , Baldi Feliciano , Giordano Antonio , Testa Joseph R. , Paggi Marco G.
Published: April 2011

Cyclin T2a was recently identified as one of the regulatory subunits of the cdk–cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9–cyclin T2a complex in this disease.
Immunohistochemical Detection of Interferon-γ: Fake or Fact?
Authors: van der Loos Chris M. , Houtkamp Mischa A. , de Boer Onno J. , Teeling Peter , van der Wal Allard C. , Becker Anton E.
Published: April 2011

Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNγ immunohistochemistry on tissue sections with a large panel of anti-IFNγ antibodies. Thirteen different commercially available anti-IFNγ antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin–biotin–peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNγ-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNγ antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNγ immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells ( n = 8), endothelial cells ( n = 4), extracellular matrix ( n = 4), and CD138+ plasma cells ( n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNIFNγ-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNγ antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNγ-immunohistochemistry must be interpreted with great caution.
Expression of the Protein Tyrosine Phosphatase-like Protein IA-2 During Pancreatic Islet Development
Authors: Roberts Caroline , Roberts Graham A. , Löbner Kristian , Bearzatto Massimo , Clark Anne , Bonifacio Ezio , Christie Michael R.
Published: April 2011

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.
Cellular Localization of Group IIA Phospholipase A2 in Rats
Authors: Yoshikawa Toshiyuki , Naruse Satoru , Kitagawa Motoji , Ishiguro Hiroshi , Nagahama Masato , Yasuda Etsuko , Semba Reiji , Tanaka Minoru , Nomura Kohji , Hayakawa Tetsuo
Published: April 2011

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA2 isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.
Plasmacytoma with Aberrant Expression of Myeloid Markers, T-cell Markers, and Cytokeratin
Authors: Shin Jung S. , Stopyra Gary A. , Warhol Michael J. , Multhaupt Hinke A.B.
Published: April 2011

Plasmacytomas are localized neoplastic proliferations of monoclonal plasma cells. When multifocal, the process is referred to as multiple myeloma. These lesions exhibit a pattern of antigen expression and cytomorphology that usually leads to a ready diagnosis. However, potentially troublesome variations in immunophenotype occur. We describe a case of a plasmacytoma from a patient who presented with sudden onset of pain and a lytic lesion of the left proximal humerus. Hematoxylin and eosin-stained sections showed a lymphoproliferative lesion composed of large lymphoid cells, some with plasmacytoid and immunoblastic features. The lesion also showed significant mitotic activity. Immunohistochemical staining was positive for CD45 (LCA), CD56 (N-CAM), CD43 (MT1), and cytokeratin CAM5.2. There was also clonal staining for λ light chains. In addition, flow cytometric analysis showed positivity for myeloid markers such as CD13, CD33, CD38, and CD138. Significant negative markers include CD20 (L26), CD45RO (UCHL-1), and CD79α. The unusual phenotypic features of this plasmacytoma illustrate potential diagnostic pitfalls. It is important to fully study such lesions to correctly classify them, because this has significant impact on prognosis and management.
Monitoring Signal Transduction in Cancer: From Chips to FISH
Authors: Lersch Robert A. , Fung Jingly , Hsieh H.-Ben , Smida Jan , Weier Heinz-Ulrich G.
Published: April 2011

The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH.
Effect of the Storage Period of Paraffin Sections on the Detection of mRNAs by In Situ Hybridization
Authors: Lisowski A.R. , English M.L. , Opsahl A.C. , Bunch R.T. , Blomme E.A.G.
Published: April 2011

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with 33P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.
Immunohistochemistry of Omega Class Glutathione S-Transferase in Human Tissues
Authors: Yin Zhan-Li , Dahlstrom Jane E. , Le Couteur David G. , Board Philip G.
Published: April 2011

Omega class glutathione transferase (GSTO) has been recently described in a number of mammalian species. We used immunohistochemistry to determine the cellular and tissue distribution of GSTO1–1 in humans. Expression of GSTO1–1 was abundant in a wide range of normal tissues, particularly liver, macrophages, glial cells, and endocrine cells. We also found nuclear staining in several types of cells, including glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid. These observations and the known activity of GSTO1–1 suggest biological functions that are not shared with other GSTs. (J Histochem Cytochem 49:983–987, 2001)
Immunolocalization of NA+,K+-ATPase in the Branchial Cavity During the Early Development of the European Lobster Homarus Gammarus (Crustacea, Decapoda)
Authors: Lignot Jean-Hervé , Charmantier Guy
Published: April 2011

We examined the ontogeny of the osmoregulatory sites of the branchial cavity in embryonic and early postembryonic stages of the European lobster Homarus gammarus through transmission electron microscopy, immunofluorescence microscopy, and immunogold electron microscopy using a monoclonal antibody IgGα5 raised against the avian α-subunit of the Na+,K+-ATPase. In mid-late embryos, Na+,K+-ATPase was located along the pleurites and within the epipodite buds. In late embryos just before hatching, the enzyme was confined to the epipodite epithelia. After hatching, slight differentiations of ionocytes occured in the epipodites of larval stages. Na+,K+-ATPase was also located in the ionocytes of the epipodites of larvae exposed to seawater (35.0‰) and to dilute seawater (22.1 ‰). After metamorphosis, the inner-side branchiostegite epithelium appeared as an additional site of enzyme location in postlarvae held in dilute seawater. Within the ionocytes, Na+,K+-ATP-ase was mostly located along the basolateral infoldings. These observations are discussed in relation to the physiological shift from osmoconforming larvae to slightly hyper-regulating (in dilute seawater) postmetamorphic stages. The acquisition of the ability to hyper-osmo-regulate probably originates from the differentiation, on the epipodites and mainly along the branchiostegites, of ionocytes that are the site of ion pumping as evidenced by the location of Na+,K+-ATPase. (J Histochem Cytochem 49:1013–1023, 2001)