bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Apoptotic Cells by Formamide-induced DNA Denaturation in Condensed Chromatin
Authors: Frankfurt Oskar S. , Krishan Awtar
Published: April 2011

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide–MAb assay for the apoptotic cells. However, TUNEL stained 90–100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA–histone interactions. Importantly, the formamide–MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.
Expression and Distribution of Tartrate-resistant Purple Acid Phosphatase in the Rat Nervous System
Authors: Lång Pernilla , Schultzberg Marianne , Andersson Göran
Published: April 2011

Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte–macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10–20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC50 for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in α-motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.
Distribution of Key Enzymes of Branched-chain Amino Acid Metabolism in Glial and Neuronal Cells in Culture
Authors: Bixel M. Gabriele , Shimomura Yoshiharu , Hutson Susan M. , Hamprecht Bernd
Published: April 2011

Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the α-keto acid products of BCAT catalyzed by the branched-chain α-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization.
Quantitation of AgNORs by Flow Versus Image Cytometry
Authors: Jacquet Bernard , Canet Véronique , Giroud Françoise , Montmasson Marie-Paule , Brugal Gérard
Published: April 2011

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase–Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.
Calmodulin Distribution and the Actomyosin Cytoskeleton in Toxoplasma gondii
Authors: Pezzella–D'Alessandro Nathalie , Le Moal Hervé , Bonhomme Annie , Valere Audrey , Klein Christophe , Gomez–Marin Jorge , Pinon Jean-Michel
Published: April 2011

The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actin–myosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca2+-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosin-CaM complex and the steps of a signaling pathway for parasite motility.
MyoD and Myogenin Expression Patterns in Cultures of Fetal and Adult Chicken Myoblasts
Authors: Yablonka–Reuveni Zipora , Paterson Bruce M.
Published: April 2011

Isolated chicken myoblasts had previously been utilized in many studies aiming at understanding the emergence and regulation of the adult myogenic precursors (satellite cells). However, in recent years only a small number of chicken satellite cell studies have been published compared to the increasing number of studies with rodent satellite cells. In large part this is due to the lack of markers for tracing avian myogenic cells before they become terminally differentiated and express muscle-specific structural proteins. We previously demonstrated that myoblasts isolated from fetal and adult chicken muscle display distinct schedules of myosin heavy-chain isoform expression in culture. We further showed that myoblasts isolated from newly hatched and young chickens already possess the adult myoblast phenotype. In this article, we report on the use of polyclonal antibodies against the chicken myogenic regulatory factor proteins MyoD and myogenin for monitoring fetal and adult chicken myoblasts as they progress from proliferation to differentiation in culture. Fetal-type myoblasts were isolated from 11-day-old embryos and adult-type myoblasts were isolated from 3-week-old chickens. We conclude that fetal myoblasts express both MyoD and myogenin within the first day in culture and rapidly transit into the differentiated myosin-expressing state. In contrast, adult myoblasts are essentially negative for MyoD and myogenin by culture Day 1 and subsequently express first MyoD and then myogenin before expressing sarcomeric myosin. The delayed MyoD-to-myogenin transition in adult myoblasts is accompanied by a lag in the fusion into myotubes, compared to fetal myoblasts. We also report on the use of a commercial antibody against the myocyte enhancer factor 2A (MEF2A) to detect terminally differentiated chicken myoblasts by their MEF2+ nuclei. Collectively, the results support the hypothesis that fetal and adult myoblasts represent different phenotypic populations. The fetal myoblasts may already be destined for terminal differentiation at the time of their isolation, and the adult myoblasts may represent progenitors that reside in an earlier compartment of the myogenic lineage.
Comparative Analysis of Galectins in Primary Tumors and Tumor Metastasis in Human Pancreatic Cancer
Authors: Berberat Pascal O. , Friess Helmut , Wang Li , Zhu Zhaowen , Bley Thorsten , Frigeri Luciano , Zimmermann Arthur , Büchler Markus W.
Published: April 2011

Galectins are galactoside-binding proteins that exhibit an important function in tumor progression by promoting cancer cell invasion and metastasis formation. Using Northern blotting and Western blotting analysis, in situ hybridization (ISH), and immunohistochemistry (IHC), we studied galectin-1 and galectin-3 in tissue samples of 33 primary pancreatic cancers and in tumor metastases in comparison to 28 normal pancreases. Furthermore, the molecular findings were correlated with the clinical and histopathological parameters of the patients. Northern blotting and Western blotting analysis showed significantly higher galectin-1 and galectin-3 mRNA and protein levels in pancreatic cancer samples than in normal controls. For galectin-1, no ISH signals and immunoreactivity were observed in acinar or ductal cells in the normal pancreas and in pancreatic cancer cells, whereas fibroblasts and extracellular matrix cells around the cancer mass exhibited strong mRNA signals and immunoreactivity. Galectin-3 mRNA signals and immunoreactivity were strongly present in most pancreatic cancer cells, whereas in the normal controls only faint ISH and IHC signals were seen in some ductal cells. Metastatic pancreatic cancer cells exhibited moderate to strong galectin-3 immunoreactivity but were negative for galectin-1. No relationship between the galectin-1 and galectin-3 mRNA levels and the tumor stage or between the IHC staining score and the tumor stage was found. However, galectin-1 mRNA levels and the IHC staining score were significantly higher in poorly differentiated tumors compared with well/moderately differentiated tumors, whereas for galectin 3 no differences were found. The expression pattern of galectin-1 and galectin-3 in pancreatic cancer tissues indicates that galectin-1 plays a role in the desmoplastic reaction that occurrs around pancreatic cancer cells, whereas galectin-3 appears to be involved in cancer cell proliferation. High levels of galectin-3 in metastatic cancer cells suggest an impact on metastasis formation.
Oxygen Insensitivity of the Histochemical Assay of Glucose-6-phosphate Dehydrogenase Activity for the Detection of (Pre)Neoplasm in Rat Liver
Authors: Jong Jonas S.S.G. De , Frederiks Wilma M. , Noorden Cornelis J.F. Van
Published: April 2011

Oxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N-Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells.

(J Histochem Cytochem 49:565–571, 2001)
Immunohistochemical Demonstration of α1,4-N-acetylglucosaminyltransferase that Forms GlcNAcα1,4Galβ Residues in Human Gastrointestinal Mucosa
Authors: Zhang Mu Xia , Nakayama Jun , Hidaka Eiko , Kubota Seiko , Yan Jing , Ota Hiroyoshi , Fukuda Minoru
Published: April 2011

α;1,4- N-acetylglucosaminyltransferase (α4GnT) is a glycosyltransferase that mediates transfer of GlcNAc to βGal residues with α1,4-linkage, forming GlcNAcα1 → 4Galβ→R structures. In normal human tissues, glycoproteins having GlcNAcα1→4Galβ→R structures at non-reducing terminals are exclusively limited to the mucins secreted from glandular mucous cells of gastric mucosa, Brunner's gland of duodenum, and accessory gland of pancreaticobiliary tract. Recently, we have isolated a cDNA encoding human α4GnT by expression cloning. Although α4GnT plays a key role in producing this unique glycan in vitro, the actual localization of α4GnT was not determined. In this study we examined the localization of α4GnT in various human tissues, including gastrointestinal mucosa, using a newly developed antibody against human α4GnT. The specificity of the antibody was confirmed by analyses of human gastric adenocarcinoma AGS cells transfected by α4GnT cDNA. Expression of α4GnT was largely associated with the Golgi region of mucous cells that produce the mucous glycoproteins having GlcNAcα1→4Galβ→R, such as the glandular mucous cells of stomach and Brunner's gland. An immunoprecipitation experiment disclosed that two distinct mucin proteins, MUC5AC and MUC6 present in gastric mucin, carried the GlcNAcα1→4Galβ→R structures. These results indicate that α4GnT is critical to form the mucous glycoproteins having GlcNAcα1→4Galβ→R on MUC6 and MUC5AC in vivo.

(J Histochem Cytochem 49:587–596, 2001)
Immunohistochemical Localization of Activated EGF Receptor in Human Eccrine and Apocrine Sweat Glands
Authors: Saga Kenji , Jimbow Kowichi
Published: April 2011

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF–EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597–601, 2001)
The Role of 15-lipoxygenase in Disruption of the Peroxisomal Membrane and in Programmed Degradation of Peroxisomes in Normal Rat Liver
Authors: Yokota Sadaki , Oda Toshiaki , Fahimi H. Dariush
Published: April 2011

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES–KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl2, 15 mM NaN3, and 0.2 M sucrose, in presence or absence of 0.5–0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes.

(J Histochem Cytochem 49:613–621, 2001)
Immunocytochemical Localization of Secretory Phospholipase A2-like Protein in the Pituitary Gland and Surrounding Tissue of the Bullfrog, Rana catesbeiana
Authors: Yaoi Yuichi , Kikuyama Sakae , Hayashi Hiroaki , Hanaoka Yoichi , Sakai Makoto , Tanaka Shigeyasu
Published: April 2011

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A2 (PLA2) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA2-like protein were observed predominately in these secretory granules. These findings support the view that this PLA2-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin.

(J Histochem Cytochem 49:631–637, 2001)
Differential Expression of Estradiol Receptors Alpha and Beta by Gonadotropes During the Estrous Cycle
Authors: Childs Gwen V. , Unabia Geda , Komak Spogmai
Published: April 2011

This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ERα or β antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) β-subunits was done on freshly dispersed pituitary cells. The lowest expression of ERα and β was seen in estrus (23% and 12%, respectively). Expression increased to 42–54% of pituitary cells by diestrus. In males, cells with ERα or β were 37% or 20% of the population, respectively. ERα or β and gonadotropin antigens were in 6–9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ERα or β with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ERα and ERβ is a part of preovulatory differentiation of pituitary gonadotropes. (J Histochem Cytochem 49:665–666, 2001)
Defining a Molecularly Normal Colon
Authors: Murphy Kelley J. , Nielson Kandice R. , Albertine Kurt H.
Published: April 2011

As techniques evolve that allow molecular characterization of disease processes such as cancer, definition of “normal” at a molecular level becomes increasingly important. Increasingly large numbers of mutations are found at the genomic level, but whether all of those mutations contribute to the malignant state of a carcinoma cell is not clear. Without knowledge of what constitutes normality on the proteomic level in an organ or cell, we cannot determine what genomic changes are physiologically important. Traditionally, colon cancer is identified and classified by histological criteria. Margins of the colon are defined as “grossly uninvolved” when the histology is indistinguishable from that of normal (free from disease) colon. By using molecular pathology techniques and working backward from colon adenocarcinoma to hypoplastic polyps to presumably normal mucosa, we defined some of those protein differences. Our results may provide a molecular basis for identifying tumor formation and progression in situ.

(J Histochem Cytochem 49:667–668, 2001)
Monitoring Signal Transduction in Cancer: Tyrosine Kinase Gene Expression Profiling
Authors: Weier Heinz-Ulrich G. , Zitzelsberger Horst F. , Hsieh H.-Ben , Sun Melita V. , Wong Mariwil , Lersch Robert A. , Yaswen Paul , Smida Jan , Kuschnick Christine , Clark Orlo H.
Published: April 2011

Abnormal expression of tyrosine kinase (TK) genes is common in tumors, in which it is believed to alter cell growth and response to external stimuli such as growth factors and hormones. Although the etiology and pathogenesis of carcinomas of the thyroid or breast remain unclear, there is evidence that the expression of TK genes, such as receptor tyrosine kinases, or mitogen-activated protein kinases, is dysregulated in these tumors, and that overexpression of particular TK genes due to gene amplification, changes in gene regulation, or structural alterations leads to oncogenic transformation of epithelial cells. We developed a rapid scheme to measure semiquantitatively the expression levels of 50–100 TK genes. Our assay is based on RT-PCR with mixed based primers that anneal to conserved regions in the catalytic domain of TK genes to generate gene-specific fragments. PCR products are then labeled by random priming and hybridized to DNA microarrays carrying known TK gene targets. Inclusion of differently labeled fragments from reference or normal cells allows identification of TK genes that show altered expression levels during malignant transformation or tumor progression. Examples demonstrate how this innovative assay might help to define new markers for tumor progression and potential targets for disease intervention. (J Histochem Cytochem 49:673–674, 2001)
Pattern of Expression of Cyclin T1 in Human Tissues
Authors: De Luca Antonio , Russo Patrizia , Severino Anna , Baldi Alfonso , Battista Tullio , Cavallotti Ivan , De Luca Luca , Baldi Feliciano , Giordano Antonio , Paggi Marco G.
Published: April 2011

Cyclin T1 was recently identified, together with cdk9 (previously named PI-TALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue.
Androgen Regulation of the Cellular Distribution of the True Tissue Kallikrein mK1 in the Submandibular Gland of the Mouse
Authors: Kurabuchi S. , Hosoi K. , Gresik E. W.
Published: April 2011

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.
High-resolution CryoFESEM of Individual Cell Adhesion Molecules (CAMs) in the Glycocalyx of Human Platelets: Detection of P-selectin (CD62P), GPI-IX Complex (CD42a/CD42bα,bβ), and Integrin GPIIbIIIa (CD41/CD61) by Immunogold Labeling and Stereo Imaging
Authors: Erlandsen Stanley L. , Bittermann Anne Greet , White James , Leith ArDean , Marko Michael
Published: April 2011

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42bα,bβ), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution back-scattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes.
Cell Type-specific Localization of Sphingosine Kinase 1a in Human Tissues
Authors: Murate Takashi , Banno Yoshiko , T-Koizumi Keiko , Watanabe Kazuko , Mori Naoyoshi , Wada Atsushi , Igarashi Yasuyuki , Takagi Akira , Kojima Tetsuhito , Asano Haruhiko , Akao Yukihiro , Yoshida Shonen , Saito Hidehiko , Nozawa Yoshinori
Published: April 2011

Cell type-specific localization of sphingosine kinase 1a (SPHK1a) in tissues was analyzed with a rabbit polyclonal antibody against the 16 C-terminal amino acids derived from the recently reported mouse cDNA sequence of SPHK1a. This antibody (anti-SPHK1a antibody) can react specifically with SPHK1a of mouse, rat, and human tissues. Utilizing its crossreactivity to human SPHK1a, the cell-specific localization of SPHK1a in human tissues was histochemically examined. Strong positive staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the red nucleus and cerebral peduncle in the midbrain, the uriniferous tubules in the kidney, the endothelial cells in vessels of various organs, and in megakaryocytes and platelets. The lining cells of sinusoids in the liver and splenic cords in the spleen showed moderate staining. Columnar epithelia in the intestine and Leydig's cells in the testis showed weak staining patterns. In addition, TPA-treated HEL cells, a human leukemia cell line, showed a megakaryocytic phenotype accompanied with increases in immunostaining of both SPHK1a and SPHK enzyme activity, suggesting that SPHK1a may be a novel marker of megakaryocytic differentiation and that this antibody is also useful for in vitro study of differentiation models.
Variations in Dystrophin Complex in Red and White Caudal Muscles from Torpedo marmorata
Authors: Royuela Mar , Hugon Gérald , Rivier François , Fehrentz Jean Alain , Martinez Jean , Paniagua Ricardo , Mornet Dominique
Published: April 2011

We present an up-to-date study on the nature, at the protein level, of various members of the dystrophin complex at the muscle cell membrane by comparing red and white caudal muscles from Torpedo marmorata. Our investigations involved immunodetection approaches and Western blotting analysis. We determined the presence or absence of different molecules belonging to the dystrophin family complex by analyzing their localization and molecular weight. Specific antibodies directed against dystrophin, i.e., DRP2 α-dystrobrevin, β-dystroglycan, α-syntrophin, α-, β-, γ-, and δ-sarcoglycan, and sarcospan, were used. The immunofluorescence study (confocal microscopy) showed differences in positive immunoreactions at the sarcolemmal membrane in these slow-type and fast-type skeletal muscle fibers. Protein extracts from T. marmorata red and white muscles were analyzed by Western blotting and confirmed the presence of dystrophin and associated proteins at the expected molecular weights. Differences were confirmed by comparative immunoprecipitation analysis of enriched membrane preparations with anti-β-dystroglycan polyclonal antibody. These experiments revealed clear complex or non-complex formation between members of the dystrophin system, depending on the muscle type analyzed. Differences in the potential function of these various dystrophin complexes in fast or slow muscle fibers are discussed in relation to previous data obtained in corresponding mammalian tissues.
Expression of Cyclo-oxygenase-2 in Naturally Occurring Squamous Cell Carcinomas in Dogs
Authors: de Almeida Ellen Maria Pestili , Piché Caroline , Sirois Jean , Doré Monique
Published: April 2011

Squamous cell carcinoma is one of the most common cancers in humans and is also a frequently diagnosed neoplasm in dogs. Induction of cyclo-oxygenase-2 (COX-2), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in the oncogenesis of various cancers in humans, including squamous cell carcinomas. However, expression of COX-2 has not been reported in spontaneous squamous cell carcinomas of non-human species. Canine squamous cell carcinomas share several similarities with the human disease. Therefore, the objective of this study was to determine whether COX isoenzymes were expressed in naturally occurring cases of squamous cell carcinomas in dogs. Canine normal skin ( n=4) and squamous cell carcinomas ( n=40) were studied by immunohistochemistry and immunoblotting analysis using polyclonal antibodies selective for COX-1 or COX-2. COX-2 was strongly expressed by neoplastic keratinocytes in all cases of squamous cell carcinomas, whereas no COX-2 was detected in normal skin and in the nonneoplastic skin and oral mucosa included in the tumor tissue samples ( p < 0.01). Immunoblotting analysis confirmed the restricted expression of COX-2 (72,000–74,000 molecular weight doublet) in squamous cell carcinomas only. In contrast, faint COX-1 staining was found in normal skin and in squamous cell carcinomas. This study demonstrates for the first time that COX-2 is induced in canine squamous cell carcinomas, and provides a new model to investigate the role and regulation of COX-2 gene expression in naturally occurring squamous cell carcinomas.
Distribution of Biglycan and Decorin in Collateral and Cruciate Ligaments and Menisci of the Rabbit Knee Joint
Authors: Kavanagh Emma , Ashhurst Doreen E.
Published: April 2011

The small leucine-rich proteoglycans (PGs) biglycan and decorin, and their mRNAs, have been localized during neonatal development and aging (3 weeks to 2 years) of collateral and cruciate ligaments and of menisci of the rabbit knee joint. In the collateral ligaments, biglycan and decorin are found between the bundles of collagen fibers at all ages. In cruciate ligaments the PGs are primarily around the cells. In neonatal ligaments all the cells express the mRNAs for biglycan and decorin, but in the collateral ligaments the number expressing the mRNAs is reduced at 8 months. In 3–week menisci the PGs are uniformly distributed in the matrix, but by 8 months biglycan is present primarily in the central fibrocartilaginous regions, whereas decorin is found peripherally. In neonates, all the cells express the mRNAs but the number is reduced in 8-month menisci. The results illustrate the precise localizations of biglycan and decorin in healthy rabbit ligaments and menisci which, after injury, must be reproduced in the repair tissue for normal strength to be regained.
Microwave Strategy for Improving the Simultaneous Detection of Estrogen Receptor and Galanin Receptor mRNA in the Rat Hypothalamus
Authors: Mitchell Valérie , Feyereisen Karine , Bouret Sébastien , Leroy Danièle , Beauvillain Jean-Claude
Published: April 2011

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor.
Lysis of Prostate Carcinoma Cells by Trifunctional Bispecific Antibodies (αEpCAM × αCD3)
Authors: Riesenberg Rainer , Buchner Alexander , Pohla Heike , Lindhofer Horst
Published: April 2011

Bispecific monoclonal antibodies (bsAbs) are a promising immunotherapeutic option for treatment of cancer, especially in situations of minimal residual disease. The combination of an anti-CD3 and anti-tumor-associated antigen antibody redirects cytotoxic T-lymphocytes towards malignant cells. Using a trifunctional bispecific antibody against Ep-CAM × CD3, that additionally activates FcγR+ accessory cells via its Fc region, we investigated the interaction between three EpCAM+ prostate carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) of healthy donors and patients with prostate carcinoma (PC). Visualization was performed by double immunocytochemical methods and computerized sequential video microscopy. Tumor cells and PBMCs supplemented with αEpCAM × αCD3 in 16-well chamber slides resulted in lysis of tumor cells within 1–3 days without any differences between patient and healthy donor PBMCs. The characteristic necrotic way of tumor cell killing (rounding, swelling, disrupting) could be observed in computerized sequences of video frames. Simultaneously, we could not reveal any form of apoptotic signal using three different apoptotic markers (TUNEL, M30 cyto death, anti-active caspase 3). Within the first 48 hr we observed typical PBMC cluster formation with increasing cell proliferation. PBMCs surrounding the tumor cells were not dominated by CD4+, CD8+, or CD14+ cells. Lymphocytes with pore-forming perforin proteins concentrated towards the tumor target cells. Our combination of double immunocytochemical and computerized video microscopic techniques may serve as an important improvement of validity of cell–cell interaction experiments using in vitro models.
Cytokeratin 20 Immunoreactivity in Renal Oncocytomas
Authors: Stopyra Gary A. , Warhol Michael J. , Multhaupt Hinke A.B.
Published: April 2011

Cytokeratins (CKs) are a group of 20 antigenically distinct intermediate filaments, generally confined to epithelia and their neoplasms. Immunostaining for CKs, in particular coordinate staining for CK7 and CK20, has become a useful tool in diagnostic pathology. Although studies defining CK distribution in neoplasms identify 0–7.7% of renal cell carcinomas (RCCs) positive for CK20, none has described the incidence of CK20 immunopositivity in renal oncocytomas (ROs). Distinction between RCC and RO may be difficult but this distinction is clinically significant, prompting us to establish the incidence of CK20 positivity in RO. We selected fifteen surgical cases of RO from our archives and studied their immunoreactivity for CKs including CK7 and CK20; 12/15 (80%) were positive for CK20, with variation in the number of cells staining. There was also variation in the distribution of CKs within the cells, including diffuse cytoplasmic, perinuclear, and a punctate or dot-like pattern. Such punctate staining corresponds to cytoplasmic balls of intermediate filaments and has been described with CAM 5.2 in RO and CK20 in Merkel cell carcinomas. Our findings suggest that CK20 immunohistochemistry is a useful tool for distinguishing RCCs from ROs.
Differential Staining of DNA Strand Breaks in Dried Comet Assay Slides
Authors: Benítez–Bribiesca L. , Sánchez P. , Toledo J. , Peñarroja R. , Flores M. , Sosa J.
Published: April 2011

The comet assay involves embedding cells in agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. We present here a simple alternative for preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal microscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was dried and reduced to a thin film, slides were treated with RNase. Image analysis showed higher tail length, total area, and tail moment. Using confocal microscopic optical sectioning, a thickness of approximately 180 μm for wet slides and 12 μm for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation of metachromasia and orthochromasia with confocal scanning microscopy. Differences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated.
Antigen Retrieval Techniques
Authors: Shi Shan-Rong , Cote Richard J. , Taylor Clive R.
Published: April 2011

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology. (J Histochem Cytochem 49:931–937, 2001)
DNA Fiber Mapping Techniques for the Assembly of High-resolution Physical Maps
Authors: Weier Heinz-Ulrich G.
Published: April 2011

High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called “DNA fibers,” may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites. (J Histochem Cytochem 49:939–948, 2001)
Immuno-EM Localization of GFP-tagged Yolk Proteins in C. Elegans Using Microwave Fixation
Authors: Paupard Marie-Christine , Miller Agnes , Grant Barth , Hirsh David , Hall David H.
Published: April 2011

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)