bluuebunny/crossref_metadata_2025 huggingface dataset:
New Fiber Formation in the Interstitial Spaces of Rat Skeletal Muscle During Postnatal Growth
Authors: Tamaki Tetsuro , Akatsuka Akira , Yoshimura Shinichi , Roy Roland R. , Edgerton V. Reggie
Published: April 2011

The purpose of this study was to determine whether fiber hyperplasia occurs in the rat plantaris muscle during postnatal weeks 3–20. Total muscle fiber number, obtained via the nitric acid digestion method, increased by 28% during the early postnatal rapid growth phase (3–10 weeks), whereas the number of branched fibers was consistently low. Whole-muscle mitotic activity and amino acid uptake levels showed an inverse relationship to the increase in total fiber number. The expression of MyoD mRNA (RT-PCR) levels decreased from 3 to 20 weeks of age, as did the detection of anti-BrdU- and MyoD-positive cells in histological sections. Immunohistochemical staining patterns for MyoD, myogenin, or developmental myosin heavy chain on sections stained for laminin (identification of the basal lamina) and electron micrographs clearly indicate that de novo fiber formation occurred in the interstitial spaces. Myogenic cells in the interstitial spaces were negative for the reliable specific satellite cell marker M-cadherin. In contrast, CD34 (an established marker for hematopoietic stem cells)-positive cells were located only in the interstitial spaces, and their frequency and location were similar to those of MyoD- and/or myogenin-positive cells. These findings are consistent with fiber hyperplasia occurring in the interstitial spaces of the rat plantaris muscle during the rapid postnatal growth phase. Furthermore, these data suggest that the new fibers may be formed from myogenic cells in the interstitial spaces of skeletal muscle and may express CD34 that is distinct from satellite cells.
Distribution Patterns of Ornithine Decarboxylase in Cells and Tissues: Facts, Problems, and Postulates
Authors: Schipper Raymond G. , Verhofstad Albert A.J.
Published: April 2011

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Increased polyamine levels are required for growth, differentiation, and transformation of cells. In situ detection of ODC in cells and tissues has been performed with biochemical, enzyme cytochemical, immunocytochemical, and in situ hybridization techniques. Different localization patterns at the cellular level have been described, depending on the type of cells or tissues studied. These patterns varied from exclusively cytoplasmic to both cytoplasmic and nuclear. These discrepancies can be partially explained by the (lack of) sensitivity and/or specificity of the methods used, but it is more likely that (sub)cellular localization of ODC is cell type-specific and/or depends on the physiological status (growth, differentiation, malignant transformation, apoptosis) of cells. Intracellular translocation of ODC may be a prerequisite for its regulation and function.
Interaction of Protein Phosphatase 1 Delta with Nucleolin in Human Osteoblastic Cells
Authors: Morimoto Hiroyuki , Okamura Hirohiko , Haneji Tatsuji
Published: April 2011

We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1δ) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1δ and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1δ and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1δ antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1δ and nucleolin was changed. Expression of PP1δ was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1δ was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1δ.
Endocytosis of Anti-CCK-B/Gastrin Receptor Antibody and Effect on Hepatoma Cell Lines
Authors: Stubbs Mark , Khan Korsa , Watson Susan A. , Savage Kay , Dhillon Amar P. , Caplin Martyn E.
Published: April 2011

Immunotherapy has considerable potential in the treatment of cancer. Here we report on the uptake of an antibody raised against the CCK-B/Gastrin receptor (CCK-BR) by liver embryonic and liver tumor cell lines. In all five cell lines studied, expression of CCK-BR and uptake of labeled anti-CCK-BR antibody was observed. The labeled anti-CCK-BR antibody was localized in both the cytoplasm and nucleus of cells. In addition, we found a coincidence between the uptake of the labeled antibody by cells and the occurrence of apoptosis (cell death). The results suggest that antibodies directed against CCK-BR have potential for targeting and possibly destroying tumor cells bearing the receptor.
Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization
Authors: Kenny Daryn , Shen Lu-Ping , Kolberg Janice A.
Published: April 2011

In situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.
Expression of Alternatively Spliced RNA Transcripts of Amelogenin Gene Exons 8 and 9 and Its End Products in the Rat Incisor
Authors: Baba Otto , Takahashi Nobuyuki , Terashima Tatsuo , Li Wu , DenBesten Pamela K. , Takano Yoshiro
Published: April 2011

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.
Methacarn Fixation for Genomic DNA Analysis in Microdissected, Paraffin-embedded Tissue Specimens
Authors: Uneyama Chikako , Shibutani Makoto , Masutomi Naoya , Takagi Hironori , Hirose Masao
Published: April 2011

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 × 1-mm area of cerebral cortex of a 10-μm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.
Localization of the Induced Metallothionein and DNA Damage in Rat Kidney After Gold Injection
Authors: Saito Shigeru , Kurasaki Masaaki , Hosokawa Toshiyuki , Okabe Masashi , Saito Takeshi , Fujii Yukiko , Nagashima Kazuo , Yoshida Katsumi
Published: April 2011

To clarify the relationships between DNA damage and Cu-MT and between DNA damage and Cu in kidneys of rats injected with Au, we examined the histochemical localization of DNA damage, metallothionein (MT), and the accumulated Cu in the kidneys of rats injected with Au, Cu, or Cu-MT. The immunoreactivity of MT was observed predominantly in the outer stripe of the outer medulla and the inner cortex of the Au-injected rat, and the signals of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) were observed in the cortex. Cu detected by Timm's method was mainly distributed in the cortex of the Au-injected rat. These results indicated that DNA damage could be caused by free Cu in the cortex but not by the Cu bound to MT in the outer stripe of the outer medulla. This consideration was supported by the data from rats injected with Cu and Cu-MT. Furthermore, we determined the Cu contents in three fractions (cytosol, organelle, and precipitate-containing nuclei) of the kidneys. Interestingly, most of the Cu content in the kidney of the rat injected with Au or Cu-MT was detected in the cytosol, whereas most of the Cu content in the kidney of the rat injected with Cu was detected in the nuclei-containing precipitate. These findings suggest that the DNA damage in the kidneys of rats injected with Au may be associated with Cu-binding proteins but not with Cu-MT.
Annexin 6 Immunoreactivity in Select Cell Populations in the Rat Brain
Authors: Moga Margaret M. , Zhou Dan
Published: April 2011

We examined the distribution of annexin 6 (ANX6) in rat brain with immuno-histochemistry (IHC). Several neuronal cell populations were intensely labeled with the ANX6 monoclonal antibody (MAb), including layer 5 of neocortex, the lateral septum, the lateral hypothalamic area, the red nucleus, and the Purkinje cell layer in cerebellum. Neuronal immunolabeling was localized to the nucleus and the cytosol. Darkly stained ANX6-immunoreactive glia, with the morphology characteristic of astrocytes, were abundant in the hippocampus, substantia nigra reticulata, and cerebellum. Evidence suggests that ANX6 may function in neuronal and glial calcium-dependent processes.
The Expression Pattern of Nitric Oxide-sensitive Guanylyl Cyclase in the Rat Heart Changes During Postnatal Development
Authors: Behrends Sönke , Mietens Andrea , Kempfert Jörg , Koglin Markus , Scholz Hasso , Middendorff Ralf
Published: April 2011

Nitric oxide (NO)-releasing drugs such as glyceryl trinitrate have been used in the treatment of ischemic heart disease for more than a century. Nevertheless, a detailed analysis of the expression of the NO target enzyme soluble guanylyl cyclase (sGC) in the heart is missing. The aim of the current study was to elucidate the expression, cell distribution, and activity of sGC in the rat heart during postnatal development. Using a novel antibody raised against a C-terminal peptide of the rat β1-subunit of sGC, the enzyme was demonstrated in early postnatal and adult hearts by Western blotting analyses, showing maximal expression in 10-day-old animals. Measurements of basal, NO-, and NO/YC-1-stimulated sGC activity revealed an increase of sGC activity in hearts from neonatal to 10-day-old rats, followed by a subsequent decrease in adult animals. As shown by immunohis-tochemical analysis, sGC expression was present in vascular endothelium and smooth muscle cells in neonatal heart but expression shifted to endothelial cells in adult animals. In isolated cardiomyocytes, sGC activity was not detectable under basal conditions but significant sGC activity could be detected in the presence of NO. An increase in expression during the perinatal period and changes in the cell types expressing sGC at different phases of development suggest dynamic regulation rather than constitutive expression of the NO receptor in the heart.
Effects of Different Fixatives on β-Galactosidase Activity
Authors: Ma Wenbin , Rogers Keith , Zbar Berton , Schmidt Laura
Published: April 2011

β-Galactosidase (β-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal β-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed β-Gal activity.
A Novel Quality Control Slide for Quantitative Immunohistochemistry Testing
Authors: Sompuram Seshi R. , Kodela Vani , Zhang Keming , Ramanathan Halasya , Radcliffe Gail , Falb Peter , Bogen Steven A.
Published: April 2011

We introduce a novel quality control technology that may improve intra- and interlaboratory immunohistochemistry (IHC) standardization. The technology involves the creation of standardized antibody targets that are attached to the same slides as the patient sample. After IHC staining, the targets turn the same color as the stained cells or tissue elements. Unlike current clinical practice, our proposed targets are neither cells nor tissue sections. To create reproducible standards that are available in unlimited supply, we use short constrained peptides as antibody targets. These peptides are attached directly to the glass slide. We show that these peptides simulate the portion of the native antigen to which the antibody binds. They are useful in detecting subtle changes in IHC staining efficacy. Moreover, the peptides do not degrade after deparaffinization or antigen retrieval treatments. This technology may be valuable in creating nationally standardized controls to quantify IHC analytical variability.
NTPDase1 and NTPDase2 Immunolocalization in Mouse Cochlea: Implications for Regulation of P2 Receptor Signaling
Authors: Vlajkovic Srdjan M. , Thorne Peter R. , Sévigny Jean , Robson Simon C. , Housley Gary D.
Published: April 2011

Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters’ cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.
Localization of Transcriptional Co-activator CBP in the Ameloblasts and the Other Enamel Organ-derived Cells of the Rat Incisor
Authors: Nishikawa Sumio
Published: April 2011

CREB-binding protein (CBP) was examined in ameloblasts and in other enamel organ-derived cells of the rat incisor, using Western blotting analysis and immunocytochemistry by specific antibodies. Western blotting of labial tissues, including ameloblasts of the incisors, detected a single band with a molecular weight equivalent to the reported value of CBP. In immunocytochemistry, CBP was localized in ameloblast nuclei in the maturation zone but not in the secretion and transition zones. The nuclei of the other enamel organ-derived cells were also positive. Because this protein is suggested to take part in c-Jun-mediated transcription, the present study and the results of a previous report showing c-Jun localization in the nuclei of enamel organ-derived cells suggest that the enamel organ-derived cells, including maturation ameloblasts, undergo active transcriptional regulation.
Expression of Hepatocyte Growth Factor mRNA in Rat Liver Cirrhosis Induced by N-nitrosodimethylamine as Evidenced by In Situ RT-PCR
Authors: Hata Junko , Ikeda Etsuko , Uno Hiroshi , Asano Satoshi
Published: April 2011

Hepatocyte growth factor (HGF) is a potent inducer of hepatocyte proliferation and is expressed during liver failure. In this study we used the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect HGF mRNA expression in normal rat livers and cirrhotic rat livers induced by treatment with N-nitrosodimethylamine (DMN). In normal control livers, in situ RT-PCR detected HGF mRNA expression in Ito cells and Kupffer cells, both of which showed rounded morphologies. However, in the cirrhotic livers induced by DMN, HGF mRNA-positive cells were spindle-shaped and surrounded the hepatocytes located around the sinusoids. These cells appeared to be sinusoidal endothelial cells as well as Ito and Kupffer cells. Because it has been suggested that HGF expression is related to transforming growth factor-β (TGF-β) levels that may play an essential role in disease progression in cirrhotic livers, TGF-β mRNA expression in normal and cirrhotic livers was also compared using in situ RT-PCR. Our results confirmed that expression of TGF-β mRNA co-localized with HGF mRNA expression in the cirrhotic liver.
Immunohistochemical Recognition of Human Follicular Dendritic Cells (FDCs) in Routinely Processed Paraffin Sections
Authors: Maeda Kunihiko , Matsuda Mikio , Suzuki Hitoshi , Saitoh Hito-aki
Published: April 2011

A number of monoclonal antibodies (MAbs) that recognize human follicular dendritic cells (FDCs) have been identified. Although some of them have already been applied individually in routine immunolabeling using formalin-fixed paraffin sections for diagnostic and experimental purposes, many antibodies are still employed only for immunolabeling using cryostat sections or particularly processed sections because they have been thought unsuitable for routine sections. A comprehensive examination re-evaluating their suitability in paraffin sections has not been reported. Accordingly, there is limited ability to examine the immunopathological contribution or diagnostic value of FDCs using routinely processed specimens or archived materials. In this study a broad panel of antibodies was systematically applied to the immunolabeling of paraffin sections of reactive tonsils or lymph nodes, in combination with advanced antigen retrieval (AR) techniques. Several antibodies, including Ki-M4p, X-11, 12B1, CNA.42, 1F8/BU32 (anti-CD21), BU38/1B12 (anti-CD23), Ber-MAC-DRC/To5 (anti-CD35), 1.4C3 (anti-CD106), NGFR5 (anti-nerve growth factor receptor p75), IIH6 (anti-CD55), 55K-2 (anti-fascin), and anti-S100 protein α-chain, were found to label FDCs in routine sections when combined with suitable AR techniques. Our results are easily adaptable for routine practice and provided useful suggestions concerning the immunopathological behavior and diversity of the particular cells.
Chromogranin A in the Human Gastrointestinal Tract: An Immunocytochemical Study with Region-specific Antibodies
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 various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411–424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 17–38 and 176–195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.
Angioarchitecture of Primary Oral Malignant Melanomas
Authors: Lee You-Jin , Nagai Noriyuki , Siar Chong-Huat , Nakano Keisuke , Nagatsuka Hitoshi , Tsujigiwa Hidetsugu , Roan Cheng-Hsiung , Gunduz Mehmet
Published: April 2011

Angiogenesis is an essential process in the progression of malignant tumors. However, little is known of the angioarchitecture in primary oral malignant melanoma. We sought to determine this by the use of periodic acid-Schiff (PAS) stain, endothelial markers (CD34, CD105) and laminin, and by transmission electron microscopy in two cases. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and these stained positively for PAS, laminin, and endothelial markers. Mosaic and tumorlined vessels were infrequently encountered. Most PAS-positive patterned networks and loops ultrastructurally represented intratumor microhemorrhages that probably arose secondary to tumor vessel leakiness. Vascular channels of the vasculogenic mimicry type were rare. They stained for laminin but not for endothelial markers.
Neuronal Developmental Marker FORSE-1 Identifies a Putative Progenitor of the Pulmonary Neuroendocrine Cell Lineage During Lung Development
Authors: Pan Jie , Yeger Herman , Cutz Ernest
Published: April 2011

The FORSE-1 ( forebrain-surface-embryonic) monoclonal antibody (MAb) recognizes a carbohydrate cell surface epitope related to the Lewis-X (LeX) and stage-specific embryonic antigens (SSEAs). In the developing CNS, the FORSE-1 epitope is believed to serve as a marker of progenitor cells. We studied the expression of the FORSE-1 epitope in pulmonary neuroendocrine cells (PNECs) and related neuroepithelial bodies (NEBs), cell types implicated in paracrine regulation of lung development. We used dual immunolabeling to identify PNECs/NEBs in tissue sections from developing rabbit fetal lungs and corresponding primary lung cell cultures. During the early stage (E16), the FORSE-1 MAb labeled primitive airway epithelium, whereas serotonin (5HT) immunoreactivity, a marker of PNEC/NEB differentiation, was negative. After E18, FORSE-1 labeling became restricted to PNECs and NEBs, identified by co-expression with 5HT, then decreased coincident with an increase in 5HT. Expression of the FORSE-1 epitope correlated inversely with 5HT expression in PNEC/NEB cells. FORSE-1 immunoreactivity correlated with cell proliferation assessed by BrdU labeling. Downregulation of the FORSE-1 epitope correlated with maturation of PNECs/NEBs. The presence of few FORSE-1/5HT-positive cells in postnatal lung suggests retention of progenitors. The FORSE-1 epitope was associated with a high molecular weight (286 kD) glycoprotein that decreased with increasing gestational age, as demonstrated by immunoblotting. Overall expression of SSEA-1, −3, and −4 antigens was similar to FORSE-1/5HT, although the former was preferentially localized to neurite-like processes. Because the role of the FORSE-1 epitope in the CNS probably involves cell adhesion and differentiation, we propose a similar function in developing lung. The demonstration of LeX/SSEA antigen expression in the PNEC/NEB cell lineage underscores the importance of these cells in developing lung. Furthermore, the FORSE-1 antigen may identify committed progenitors of the PNEC/NEB cell system.
Structural Integrity of the Golgi Stack Is Essential for Normal Secretory Functions of Rat Parotid Acinar Cells: Effects of Brefeldin A and Okadaic Acid
Authors: Tamaki Hideaki , Yamashina Shohei
Published: April 2011

We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (β-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and β-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules.
Distinct Distribution of Laminin and Its Integrin Receptors in the Pancreas
Authors: Jiang Fang-Xu , Naselli Gaetano , Harrison Leonard C.
Published: April 2011

Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin β1- and γ1-chains, the α1-chain, unique to laminin-1, was not detected. Laminin-10 (α5β1γ1) was expressed in acinar tissue, whereas laminins-2 (α2β1γ1) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin β1 and γ1, whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits α6 and α3 were detected in acinar cells and duct epithelial cells, but α6 was absent in islet cells. Integrin α6β4 was detected only in duct cells, α6β1 in both acinar and ductal cells, and α3β1 in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.
Gender- and Age-dependent Changes in Kidney Androgen Protein mRNA Expression in a Knockout Mouse Model for Nephrolithiasis
Authors: Tzortzaki Eleni G. , Glass Dayna , Yang Min , Evan Andrew P. , Bledsoe Sharon B. , Stambrook Peter J. , Sahota Amrik , Tischfield Jay A.
Published: April 2011

Kidney androgen-regulated protein (Kap) is the most abundant protein in the mouse kidney, but its function is unknown. We previously observed a significant decrease in Kap mRNA expression in whole kidney tissue from male mice with adenine phosphoribosyltransferase (APRT) deficiency and 2,8-dihydroxyadenine (DHA) nephrolithiasis. The disease phenotype is more severe in male mice and is age-dependent. To identify the cellular basis for differential Kap expression, we used in situ hybridization (ISH) and reverse transcription-polymerase chain reaction ISH (RT-PCR ISH) to identify the cell types expressing this mRNA in paraffin-embedded kidney sections. In 1-month-old wild-type male mice, Kap was detected primarily in S3 proximal tubule segments, but expression was very low in female mice. In 1-month-old APRT-deficient male mice, Kap expression was decreased significantly and was undetectable in female mice. Kap mRNA was not detected in 3- or 6-month-old mice using our standard ISH protocol, but we observed intense cytoplasmic staining in S3 proximal tubules in wild-type male mice of these age groups using an improved RT-PCR ISH procedure. Our studies demonstrate age-, gender-, and APRT genotype-dependent changes in Kap mRNA expression in mouse kidney. Kap expression is under multihormonal control, and hormonal changes in DHA-induced nephrolithiasis may account for the decreased Kap expression in APRT-deficient mice.
Effect of Fixation Period on HER2/neu Gene Amplification Detected by Fluorescence In Situ Hybridization in Invasive Breast Carcinoma
Authors: Selvarajan Sathiyamoorthy , Bay Boon-Huat , Choo Andrew , Chuah Khoon-Leong , Sivaswaren Christina Rudduck , Tien Sim-Leng , Wong Chow-Yin , Tan Puay-Hoon
Published: April 2011

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.
Suspension (S)-FISH, a New Technique for Interphase Nuclei
Authors: Steinhaeuser Ulf , Starke Heike , Nietzel Angela , Lindenau Joerg , Ullmann Peter , Claussen Uwe , Liehr Thomas
Published: April 2011

We describe a versatile method for performing fluorescence in situ hybridization (FISH) in suspension instead of on a slide as usually done. This so-called suspension-FISH (S-FISH) opens new possibilities for the analysis of shape and functions of the human interphase nucleus. The procedure is described and the first results using this approach are presented.
The Antigen-Antibody Reaction in Immunohistochemistry
Authors: Montero Claudio
Published: April 2011

The problems of major concern in immunohistochemical practice are discussed in the following order: (a) the mechanism of the Ag-Ab reaction in fixed tissue as opposed to the in vitro reaction; (b) the chemistry of fixation and its influence on the final result of the immunohistochemical reaction; (c) the various procedures used for antigen retrieval in formaldehyde-fixed tissue; and (d) the consideration of the possible mechanism underlying heat-induced antigen retrieval. Suggestions for further work to attempt a clarification of the mechanism involved in the Ag-Ab reaction in immunohistochemistry resorting to existing histochemical methods for the demonstration of protein side groups are presented, together with some examples already published.
Localization of Messenger Ribonucleic Acids for Somatostatin Receptor Subtypes (sstr1–5) in the Rat Adrenal Gland
Authors: O'Carroll Anne-Marie
Published: April 2011

Somatostatin (somatotropin-release inhibitory factor, SRIF) exerts multiple inhibitory actions throughout the central nervous system and the periphery by binding to specific membrane-bound SRIF receptors (sstrs) of which five subtypes (sstr1–5) have now been identified. Individual sstr subtypes have been suggested to mediate selective biological actions of SRIF. Although the adrenal gland is a known target of SRIF action, the sstr subtypes involved in its actions are unclear. This study examined the expression of sstr1–5 in rat adrenal gland by RT-PCR analysis and in situ hybridization (ISH) histochemistry. Using RT-PCR expression combined with Southern blotting, sstr1, −2, −4, and −5 mRNAs were shown in the adrenal gland. ISH histochemistry revealed strong expression of sstr2 mRNA alone localized to the zona glomerulosa of the adrenal cortex and moderate labeling in scattered cells of the adrenal medulla, indicating a possible role for sstr2 in mediating SRIF physiology in this tissue by altering adrenal aldosterone and catecholamine secretion. These data also point to potential roles for sstr subtypes sstr1, −4, and −5 in the adrenal gland.
p63 Is Expressed in Basal and Myoepithelial Cells of Human Normal and Tumor Salivary Gland Tissues
Authors: Bilal Hadi , Handra-Luca Adriana , Bertrand Jacques-Charles , Fouret Pierre J.
Published: April 2011

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.
Morphological Insights into the Origin of Glomerular Endothelial and Mesangial Cells and Their Precursors
Authors: Ricono Jill M. , Xu Yi-Chun , Arar Mazen , Jin Dong-chan , Barnes Jeffrey L. , Abboud Hanna E.
Published: April 2011

Glomerular endothelial and mesangial cells may originate from the metanephric mesenchyme. We used the MAb Thy1.1, a mesangial cell marker in the adult rat kidney, and rat endothelial cell markers MAb RECA-1, MAb PECAM-1 (CD31), and MAb Flk-1 as potential markers to characterize the spatial and temporal distribution of mesangial and endothelial cell precursors during nephrogenesis in the rat. At early stages of glomerulogenesis, RECA-1- and Thy1.1-positive cells were detected in the metanephric blastema at 14 days post conception (dpc) embryos and 15 dpc, respectively, with Thy1.1 expression in cells surrounding the ureteric bud. At 17 and 18 dpc, both RECA-1- and Thy1.1-positive cells were found in the cleft of the S-shaped bodies and in the capillary loops of maturing glomeruli. Double staining for BrdU, a marker of proliferation, and for RECA-1 or BrdU and Thy1.1 also localize in the cleft of S-shaped bodies and in glomerular capillary loops at later stages of development. PDGFRβ co-localizes in cells expressing endothelial or mesangial markers. The data suggest that endothelial and mesangial cell precursors share common markers during the course of glomerulogenesis and that full differentiation of these cells occurs at late stages of glomerular maturation. Thy1.1- and RECA-1-positive cells may be derived from the metanephric blastemal cells at early stages of kidney development. A sub-population of these Thy1.1- or RECA-1-positive cells may be precursors that can migrate into the cleft of comma and S-shaped bodies and proliferate in situ to form glomerular capillary tufts.
Expression of Human Pendrin in Diseased Thyroids
Authors: Kondo Tetsuo , Nakamura Nobuki , Suzuki Koichi , Murata Shin-ichi , Muramatsu Akira , Kawaoi Akira , Katoh Ryohei
Published: April 2011

We examined pendrin expression in various diseased thyroid tissues by immunohistochemistry (IHC) using antiserum raised against human pendrin and by real-time quantitative RT-PCR. In normal thyroids the antiserum reacted with the apical membrane of follicular cells and its immunoreactivity was faint. In Graves’ thyroids, the IHC expression of pendrin appeared in a pattern similar to that of normal thyroids but it was more extensive and stronger, especially in areas showing marked proliferation of follicular cells. The immunoreactivities of pendrin in nodular goiters varied from case to case. In follicular adenomas, pendrin was localized in the follicle-forming parts of the tumor but was negative in trabecular parts. Pendrin was negative in all follicular carcinomas, papillary carcinomas, and in one case of medullary carcinoma. In quantitive mRNA analysis, the relative values of pendrin mRNA were significantly low in papillary carcinoma ( p<0.01), whereas the values in other diseased thyroids were not significantly different from those in normal thyroids. These results suggest that pendrin may play a role in thyroid hormone production as the apical porter of chloride/iodide and investigation of pendrin leads to a better understanding of functional aspects of the iodine transportation system in thyroid diseases.
An Antigen Retrieval Method Using an Alkaline Solution Allows Immunoelectron Microscopic Identification of Secretory Granules in Conventional Epoxy-embedded Tissue Sections
Authors: Yano Shinji , Kashima Kenji , Daa Tsutomu , Urabe Shogo , Tsuji Koichi , Nakayama Iwao , Yokoyama Shigeo
Published: April 2011

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO4, and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 ± 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 ± 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.
Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Accurately Measures Receptor Number
Authors: Matkowskyj Kristina A. , Cox Randal , Jensen Robert T. , Benya Richard V.
Published: April 2011

We previously demonstrated that quantitative immunohistochemistry (Q-IHC) performed by measuring the cumulative signal strength of the digital file encoding an image can be used to determine the absolute amount of chromogen present per pixel. We now show that Q-IHC so performed can be used to accurately determine the amount of peptide hormone receptor of interest in archived tissues. To do this we transfected Balb 3T3 fibroblasts with the cDNA encoding the human receptor for gastrin-releasing peptide (GRP), and selected six cell lines stably expressing between 102 and 106 receptors/cell. These cell lines were fixed in formalin, embedded in paraffin, and treated with antipeptide antibodies against the GRP receptor, followed by DAB chromogen to identify bound antibody. Images were acquired using a 4.9 million pixel digital scanning 24-bit RGB camera, saved in TIFF format, and used for subsequent analysis. Q-IHC was performed after digitally dissecting out the relevant portion of the image for analysis, and processing using a program written in C (available at http://www.uic.edu/com/dom/gastro/Freedownloads.html ). Under the conditions defined here, chromogen quantity as determined by Q-IHC tightly correlated with GRP receptor number (r2=0.867) in these cell lines. Using the conversion factor identified as a result of these studies, we then determined GRP receptor number on eight randomly selected, archived human colon cancers. Overall GRP receptor expression in colon cancer depended on the degree to which cells within any particular tumor were differentiated, with well-differentiated cells expressing the greatest numbers of receptors (∼55,000 ± 10,000 sites/cell). These studies indicate that Q-IHC can be used to determine receptor quantity in archived tissues and other samples of limited quantity.
Lectin Histochemistry Shows Fucosylated Glycoconjugates in the Primordial Germ Cells of Xenopus Embryos
Authors: Alonso Edurne , Sáez Francisco J. , Madrid Juan F. , Hernández Francisco
Published: April 2011

Previous works have shown that glycoconjugates with terminal fucose (Fuc) are located in the primordial germ cells (PGCs) of some mammals and might play a role in the migration and adhesion processes during development. The aim of this work was to identify the terminal Fuc moieties of Xenopus PGCs by means of three Fuc-binding lectins: from asparagus pea (LTA), gorse seed (UEA-I), and orange peel fungus (AAA). The histochemical procedures were also carried out after deglycosylation pretreatments: β-elimination with NaOH to remove O-linked oligosaccharides; incubation with PNGase F to remove N-linked carbohydrate chains; and incubation with α(1,2)- and α(1,6)-fucosidase. The PGCs were always negative for LTA and UEA-I, two lectins that have the highest affinity for Fuc α(1,2)-linked. However, the PGCs were strongly labeled with AAA, which preferentially binds to Fuc with α(1,3) or α(1,4) linkages and to Fuc α(1,6)-linked to the proximal N-acetylglucosamine. There was fainter labeling with AAA when the sections were preincubated with α(1,6)-fucosidase, but the labeling remained strong when the sections were pretreated with α(1,2)fucosidase. When the β-elimination procedure was carried out, the PGC labeling with AAA was slight. If the PNGase F incubation was performed, the PGCs remained moderately positive for AAA. These data suggest that the Xenopus PGCs have Fuc moieties in O- and N-linked oligosaccharides, including Fuc α(1,6) linked to the innermost GlcNAc, and that the Fuc was not in α(1,2)-linkage.
DNA Ploidy and Markovian Analysis of Neoplastic Progression in Experimental Pancreatic Cancer
Authors: Postier Russell G. , Lerner Megan R. , Lightfoot Stan A. , Vannarath Rick , Lane Mary M. , Hanas Jay S. , Brackett Daniel J.
Published: April 2011

Computer-assisted analysis of DNA ploidy and nuclear morphology were used to elucidate changes in the cell nucleus that occur during the development of experimental pancreatic cancer. Ductal pancreatic adenocarcinoma was induced in 49 Syrian hamsters by SC injection of N-nitrosobis (2-oxopropyl) amine; twenty hamsters served as controls. Groups of animals were sacrificed every 4 weeks for 20 weeks and adjacent sections of pancreatic tissue were H&E and Feulgen-stained for light microscopy and computer assisted cytometry. Pancreatic ductal cells were classified as normal, atypical, or malignant; tissue inflammation (pancreatitis) was also noted when present. DNA ploidy and nuclear morphology evaluation (Markovian analysis) identified an atypical cell stage clearly distinguishable from either normal or malignant cells; pancreatitis preceded this atypia. The DNA ploidy histogram of these atypical cells revealed a major diploid peak and a minor aneuploid peak. The receiver operator characteristic curve areas for a logistic regression model of normal vs atypical cells was 0.94 and for atypical vs malignant was 0.98, numbers indicative of near-perfect discrimination among these three cell types. The ability to identify an atypical cell population should be useful in establishing the role of these cells in the progression of human pancreatic adenocarcinoma.
Lymphatic Network and Lymphangiogenesis in the Gastric Wall
Authors: Ji Rui-Cheng , Kato Seiji
Published: April 2011

A family of growth factors highly specific for endothelial cells was identified more than 10 years ago, in which the receptor of vascular endothelial growth factor C (VEGFR-3) is implicated in the regulation of lymphatic development and regeneration. Comparative studies on the lymphatic network and lymphangiogenesis have been done mainly using combined 5′-nucleotidase (5′-Nase) enzyme and VEGFR-3 immunohistochemical approaches in adult and fetal gastric walls. Developing lymphatic networks represented fewer blind ends and branches than mature networks in whole-mount preparations. Many circular lymphatic-like structures exhibited VEGFR-3 expression and weak 5′-Nase activity in the early embryonic stage, showing visible morphological properties in the lymphatic endothelium. These newly formed lymphatics showed an obvious accumulation in the submucosa and serosa and a variation in the intensity of VEGFR-3 binding to endothelial cells among samples. A reaction product for anti-VEGFR-3 was found on the luminal surface of endothelial cells and on the membrane of some organelles and intraluminal lymphocytes. These findings indicate that an active proliferating feature of the clustered developing lymphatics may create a favorable environment for their sprouting and growth, which may serve as a functional requirement for lymph drainage in the region.