bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal Analysis of Rat Growth Plates: Cessation of Growth with Age Despite Presence of a Physis
Authors: Roach Helmtrud I. , Mehta Gautam , Oreffo Richard O.C. , Clarke Nicholas M.P. , Cooper Cyrus
Published: April 2011

Despite the continued presence of growth plates in aged rats, longitudinal growth no longer occurs. The aims of this study were to understand the reasons for the cessation of growth. We studied the growth plates of femurs and tibiae in Wistar rats aged 62–80 weeks and compared these with the corresponding growth plates from rats aged 2–16 weeks. During skeletal growth, the heights of the plates, especially that of the hypertrophic zone, reflected the rate of bone growth. During the period of decelerating growth, it was the loss of large hydrated chondrocytes that contributed most to the overall decrease in the heights of the growth plates. In the old rats we identified four categories of growth plate morphology that were not present in the growth plates of younger rats: (a) formation of a bone band parallel to the metaphyseal edge of the growth plate, which effectively sealed that edge; (b) extensive areas of acellularity, which were resistant to resorption and/or remodeling; (c) extensive remodeling and bone formation within cellular regions of the growth plate; and (d) direct bone formation by former growth plate chondrocytes. These processes, together with a loss of synchrony across the plate, would prevent further longitudinal expansion of the growth plate despite continued sporadic proliferation of chondrocytes.
Homeobox Protein, Hmx3, in Postnatally Developing Rat Submandibular Glands
Authors: Shaw Phyllis A. , Zhang Xu , Russo Andrew F. , Amendt Brad A. , Henderson Scott , Williams Valerie
Published: April 2011

Homeobox-containing (Hox) genes play important roles in development, particularly in the development of neurons and sensory organs, and in specification of body plan. The Hmx gene family is a new class of homeobox-containing genes defined by a conserved homeobox region and a characteristic pattern of expression in the central nervous system that is more rostral than that of the Hox genes. To date, three closely related members of the Hmx family, Hmx1, Hmx2, and Hmx3, have been described. All three Hmx genes are expressed in the craniofacial region of developing embryos. Here we show, for the first time, the expression of the transcription factor Hmx3 in postnatally developing salivary glands. Hmx3 protein is expressed in a cell type-specific manner in rat salivary glands. Hmx3 is present in both the nuclei and cytoplasm of specific groups of duct cells of the submandibular, parotid, and sublingual glands. Hmx3 expression increases during postnatal development of the submandibular gland. The duct cells show increasing concentrations of Hmx3 protein with progressive development of the submandibular gland. In contrast, the acinar cells of the three salivary glands do not exhibit detectable levels of Hmx3 protein.
Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor
Authors: Jockusch Harald , Voigt Sylvana , Eberhard Daniel
Published: April 2011

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.
Role of Myosin II in Axon Outgrowth
Authors: Brown Jacquelyn , Bridgman Paul C.
Published: April 2011

The initial stages of nerve outgrowth carried out by growth cones occur in three fundamental cyclic steps. Each of these steps appears to require myosin II activity to variable degrees. The steps include the following: (a) exploration, involving extensions and retractions that are driven and controlled by the interaction of actin retrograde flow and polymerization; (b) adhesion of new extensions to the substrate, which has been shown to be mediated by complex interactions between extracellular matrix proteins, cell adhesion proteins, and the actin cytoskeleton; and (c) traction force generated during forward advance of the growth cone, resulting in the production of tension on the neurite.
Growth Cones Integrate Signaling from Multiple Guidance Cues
Authors: Dontchev Vassil D. , Letourneau Paul C.
Published: April 2011

Nerve growth factor (NGF) and semaphorin3A (Sema3A) are guidance cues found in pathways and targets of developing dorsal root ganglia (DRG) neurons. DRG growth cone motility is regulated by cytoplasmic signaling triggered by these molecules. We investigated interactions of NGF and Sema3A in modulating growth cone behaviors of axons extended from E7 chick embryo DRGs. Axons extending in collagen matrices were repelled by Sema3A released from transfected HEK293 cells. However, if an NGF-coated bead was placed adjacent to Sema3A-producing cells, axons converged at the NGF bead. Growth cones of DRGs raised in 10-9 M NGF were more resistant to Sema3A-induced collapse than when DRGs were raised in 10-11 M NGF. After overnight culture in 10-11 M NGF, 1-hr treatment with 10-9 M NGF also increased growth cone resistance to Sema3A. Pharmacological studies indicated that the activities of ROCK and PKG participate in the cytoskeletal alterations that lead to Sema3A-induced growth cone collapse, whereas PKA activity is required for NGF-mediated reduction of Sema3A-induced growth cone collapse. These results support the idea that growth cone responses to a guidance cue can be modulated by interactions involving coincident signaling by other guidance cues.
Expression of the LIM Proteins Paxillin and Hic-5 in Human Tissues
Authors: Yuminamochi Tsutomu , Yatomi Yutaka , Osada Makoto , Ohmori Tsukasa , Ishii Yoshio , Nakazawa Kumiko , Hosogaya Shigemi , Ozaki Yukio
Published: April 2011

The LIM domain is a protein-protein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation.
Intracellular Targeting of Calmodulin mRNAs in Primary Hippocampal Cells
Authors: Kortvely Elod , Varszegi Szilvia , Palfi Arpad , Gulya Karoly
Published: April 2011

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.
Differential Expression of Ribosomal Proteins in Human Normal and Neoplastic Colorectum
Authors: Kasai Hide , Nadano Daita , Hidaka Eiko , Higuchi Kayoko , Kawakubo Masatomo , Sato Taka-Aki , Nakayama Jun
Published: April 2011

Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithlia except immature mucosal cells, whereas their expression levels were significantly enchanced in colorectal cancer cells. In addition, L7 ribosomal protien was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.
Automated Selection of DAB-labeled Tissue for Immunohistochemical Quantification
Authors: Brey Eric M. , Lalani Zahid , Johnston Carol , Wong Mark , McIntire Larry V. , Duke Pauline J. , Patrick Charles W.
Published: April 2011

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.
S100 Protein Subcellular Localization During Epidermal Differentiation and Psoriasis
Authors: Broome Ann-Marie , Ryan David , Eckert Richard L.
Published: April 2011

S100 proteins are calcium-activated signaling proteins that interact with target proteins to modulate biological processes. Our present studies compare the level of expression, and cellular localization of S100A7, S100A8, S100A9, S100A10, and S100A11 in normal and psoriatic epidermis. S100A7 and S100A11 are present in the basal and spinous layers in normal epidermis. These proteins appear in the nucleus and cytoplasm in basal cells but are associated with the plasma membrane in spinous cells. S100A10 is present in basal and spinous cells, in the cytoplasm, and is associated with the plasma membrane. S100A8 and S100A9 are absent or are expressed at minimal levels in normal epidermis. In involved psoriatic tissue, S100A10 and S100A11 levels remain unchanged, whereas, S100A7, S100A8, and S100A9 are markedly overexpressed. The pattern of expression and subcellular localization of S100A7 is similar in normal and psoriatic tissue. S100A8 and S100A9 are strongly expressed in the basal and spinous layers in psoriasis-involved tissue. In addition, we demonstrate that S100A7, S100A10, and S100A11 are incorporated into detergent and reducing agent-resistant multimers, suggesting that they are in vivo trans-glutaminase substrates. S100A8 and S100A9 did not form these larger complexes. These results indicate that S100 proteins localize to the plasma membrane in differentiated keratinocytes, suggesting a role in regulating calcium-dependent, membrane-associated events. These studies also indicate, as reported previously, that S100A7, S100A8, and S100A9 expression is markedly altered in psoriasis, suggesting a role for these proteins in disease pathogenesis.
Enhanced Serine Palmitoyltransferase Expression in Proliferating Fibroblasts, Transformed Cell Lines, and Human Tumors
Authors: Carton Jill M. , Uhlinger David J. , Batheja Ameesha D. , Derian Claudia , Ho George , Argenteri Dennis , D'Andrea Michael R.
Published: April 2011

Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.
Differential Expression of Heparan Sulfate Domains in Rat Spleen
Authors: Dam Gerdy B. ten , Hafmans Theo , Veerkamp Jacques H. , Kuppevelt Toin H. van
Published: April 2011

The microarchitecture of the spleen is composed of a meshwork of reticulum cells and their matrix. Heparan sulfates (HS) are important components of this meshwork and are involved in processes such as cell adhesion, cell migration, and cytokine/growth factor binding. The expression of HS epitopes was analyzed using anti-HS antibodies. Four different staining patterns were observed, as exemplified by antibodies RB4EA12, HS4E4, AO4B08, and HS4C3. These antibodies recognize different chemical modifications in HS. In adult spleen, RB4EA12 stained only the reticular meshwork and blood vessels in the red pulp and marginal zone. HS4E4 stained blood vessel-associated basal lamina. AO4B08 and HS4C3 stained the reticular meshwork and blood vessels throughout the spleen, but only AO4B08 strongly stained smooth muscle cells and ring fibers. Interleukin-2 localized in the red pulp and marginal zone and was bound to HS. AO4B08, HS4C3, and RB4EA12 but not HS4E4 co-localized with interleukin-2. In 10-day-old spleen, HS4E4 recognized reticular fibers, which were not stained in the adult stage. Immunoelectron microscopy revealed that HS was restricted to basal laminae and reticular fibers. Taken together, data show that HS epitopes are differentially expressed in the spleen and that this may create specific extracellular environments for immunological processes.
Patterns of MUC1 Tissue Expression Defined by an Anti-MUC1 Cytoplasmic Tail Monoclonal Antibody in Breast Cancer
Authors: Croce María V. , Isla-Larrain Marina T. , Rua Carina E. , Rabassa Martín E. , Gendler Sandra J. , Segal-Eiras Amada
Published: April 2011

Our aim was to determine the pattern of expression of MUC1 mucin cytoplasmic tail (MUC1 CT) in breast carcinoma. A total of 98 invasive breast adenocarcinoma tumor samples were assayed by immunohistochemical (IHC) analysis. The pattern of reaction was classified as membrane, cytoplasmic, or mixed. Subcellular fractions were prepared after SDS-PAGE and Western blotting. The antibodies employed were anti-MUC1 CT (CT2 monoclonal antibody, MAb) and C595 MAb against the extracellular MUC1 core protein. With the CT2 MAb, IHC showed a high percentage of positive staining in 93% of specimens, with membrane staining the most common pattern observed. C595 MAb was reactive in 73% of specimens. Similar percentages of membrane and cytoplasmic staining were found, mainly in a mixed pattern. Western blotting showed different bands. With the CT2 MAb, the membrane fraction showed the most intense reaction; a strong band of reaction was detected at approximately <30 kD. With the C595 MAb, in most cases a double band at 200 kD was found. In breast epithelium, the pattern of MUC1 CT expression may constitute an indicator of MUC1 production because it does not depend on glycosylation. The pattern and extension of MUC1 CT positivity do not vary according to the histopathological subtype of the tumor.
Skeletal Muscles Express the Xenobiotic-metabolizing Enzyme Arylamine N-acetyltransferase
Authors: Rodrigues-Lima Fernando , Cooper Racquel N. , Goudeau Bertrand , Atmane Noureddine , Chamagne Anne-Marie , Butler-Browne Gillian , Sim Edith , Vicart Patrick , Dupret Jean-Marie
Published: April 2011

The human arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are enzymes responsible for the acetylation of many arylamines and hydrazines, thereby playing an important role in both detoxification and activation of many drugs and carcinogens. Both enzymes show polymorphisms but exhibit key differences in substrate selectivity and tissue expression. In the present study, reverse transcriptase-PCR, Western blotting, and immunohistochemistry were used to investigate the expression of the NATs in human skeletal muscle. Despite the presence of its mRNA, NAT2 enzyme level was below the limit of detection. In contrast, both NAT1 mRNA and enzyme were readily detected in fetal, newborn, and adult muscles. In addition, punctate cytoplasmic and perinuclear NAT1 immunostaining was observed in all tissue sections, the staining being more intense in the fetal tissue. High expression of NAT1 enzyme in fetal muscle was also suggested by Western blotting. Because skeletal muscle accounts for a large proportion of body mass, muscle NAT1 expression may contribute significantly to the total activity in the body. These results further support the involvement of skeletal muscle in the metabolism of xenobiotics.
Brain Synaptic Junctional Proteins at the Acrosome of Rat Testicular Germ Cells
Authors: Redecker Peter , Kreutz Michael R. , Bockmann Jürgen , Gundelfinger Eckart D. , Boeckers Tobias M.
Published: April 2011

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.
Quantitative Histochemical Assay for Superoxide Dismutase in Rat Brain
Authors: Viggiano Alessandro , Viggiano Davide , Viggiano Andrea , Luca Bruno De
Published: April 2011

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.
Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis
Authors: Yasuhara Shingo , Zhu Ying , Matsui Takashi , Tipirneni Naveen , Yasuhara Yoko , Kaneki Masao , Rosenzweig Anthony , Martyn J.A. Jeevendra
Published: April 2011

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.
The Distribution of Abcc6 in Normal Mouse Tissues Suggests Multiple Functions for this ABC Transporter
Authors: Beck Konstanze , Hayashi Kimiko , Nishiguchi Brian , Saux Olivier Le , Hayashi Masando , Boyd Charles D.
Published: April 2011

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.
Combined Tyramide Signal Amplification and Quantum Dots for Sensitive and Photostable Immunofluorescence Detection
Authors: Ness Jayne M. , Akhtar Rizwan S. , Latham Cecelia B. , Roth Kevin A.
Published: April 2011

Conventional immunofluorescence detection of biologically relevant proteins and antigens in tissue sections is often limited by relatively weak signals that fade rapidly on illumination. We have developed an immunohistochemical protocol that combines the sensitivity of tyramide signal amplification with the photostability of quantum dots to overcome these limitations. This simple method provides a sensitive and stable fluorescence immunohistochemical alternative to standard chromogen detection.
Cytoprotective Effects of Polyenoylphosphatidylcholine (PPC) on β-cells During Diabetic Induction by Streptozotocin
Authors: Lee Seung-Hee , Han Yu-Mi , Min Bon-Hong , Park In-Sun
Published: April 2011

Polyenoylphosphatidylcholine (PPC), a phosphatidylcholine-rich phospholipid extracted from soybean, has been reported to protect liver cells from alloxan-induced cytotoxicity. The present study aimed to investigate whether PPC protects pancreatic β-cells from the cytotoxic injury induced by streptozotocin, thus preserving insulin synthesis and secretion. β-Cells of the PPC-treated rats showed a significant reduction of cell death with lesser destruction of plasma membrane on streptozotocin insult. They demonstrated a rapid recovery of GLUT-2 expression, whereas almost irreversible depletion of membranebound GLUT-2 was seen in β-cells of the rats treated with only streptozotocin. A similar cytoprotective effect of PPC was also monitored in the PPC-pretreated MIN6 cells. These β-cells retained their ability to synthesize and secrete insulin and no alteration of glucose metabolism was detected. These results strongly suggest that PPC plays important roles not only in protecting β-cells against cytotoxicity but also in maintaining their insulin synthesis and secretion for normal glucose homeostasis.
Expression of the Two Isoforms of the Vesicular Monoamine Transporter (VMAT1 and VMAT2) in the Endocrine Pancreas and Pancreatic Endocrine Tumors
Authors: Anlauf Martin , Eissele Rolf , Schäfer Martin K.-H. , Eiden Lee E. , Arnold Rudolf , Pauser Ursula , Klöppel Günter , Weihe Eberhard
Published: April 2011

The uptake of monoamines into the secretory granules of monoamine-storing neuroendocrine cells is mediated by vesicular monoamine transporter protein 1 or 2 (VMAT1 or VMAT2). This study analyzed the expression of VMAT1 and VMAT2 in endocrine cells of normal human and monkey pancreas. The expression of VMAT1 and VMAT2 was also examined in infants with hyperinsulinemic hypoglycemia and in adults with pancreatic endocrine tumors (PETs). Using immunohistochemistry (IHC) and in situ hybridization (ISH), we demonstrated the mutually exclusive expression of VMAT1 in endocrine cells of the duct system and of VMAT2 in many cells of the islets of Langerhans. By confocal laser scanning microscopy, VMAT1-positive cells were identified as enterochromaffin (EC) cells and VMAT2-positive cells as β-cells. In PETs, VMAT1 was found exclusively in all serotonin-containing tumors. In contrast, VMAT2 expression was lost in many insulinomas, independent of their biological behavior. VMAT2 was expressed by some non-insulin-producing tumors. The mutually exclusive expression of VMAT1 in EC cells and of VMAT2 in β-cells suggests that both cell types store monoamines. Monoamine storage mediated by VMAT1 in EC cells is apparently maintained in EC cell tumors. In contrast, many insulinomas appear to lose their ability to accumulate monoamines via VMAT2.
Localization of VIP36 in the Post-Golgi Secretory Pathway Also of Rat Parotid Acinar Cells
Authors: Shimada Osamu , Hara-Kuge Sayuri , Yamashita Katsuko , Tosaka-Shimada Hisami , Yanchao Li , Einan Li , Atsumi Saoko , Ishikawa Harunori
Published: April 2011

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.
Immunohistochemical Comparison of Anti-prion Protein (PrP) Antibodies in the CNS of Mice Infected with Scrapie
Authors: Liu Wing Gee , Brown Debbie A. , Fraser Janet R.
Published: April 2011

One of the pathological changes characteristic of the transmissible spongiform encephalopathies (TSEs) is the accumulation of disease-specific PrP (PrPsc). Immunola-beling of PrPsc was compared using a panel of monoclonal and polyclonal antibodies. To determine the effects of tissue fixation on immunostaining, we performed a supplementary investigation reviewing the fixatives formol saline and periodate-lysine-paraformaldehyde (PLP). The main target sites of the antibodies were similar. However the monoclonal antibodies (MAbs) 6H4, 7A12 and 8H4 revealed targeted PrPsc labeling with no background labeling. Although 7A12 and 8H4 did not detect early PrP deposition, we propose that during the later stages of disease 7A12 and 8H4 can be used with equal effectiveness in place of 6H4. Tissues taken during the early stages of disease that had been fixed in PLP displayed more PrP immunolabeling than tissues that had undergone formol fixation. PLP fixation on 6H4-immunostained tissue revealed interweaving granular linear PrP deposits in the hippocampus. This labeling was not observed in tissue that had undergone formol fixation, suggesting that PLP fixation might enhance the sensitivity of the immuno-histochemical (IHC) detection of PrP. In the two scrapie mouse models studied here, PLP fixation and immunolabeling with the anti-PrP antibody 6H4 gave superior results.
Biochemical Characterization and Immunocytochemical Localization of EM66, a Novel Peptide Derived from Secretogranin II, in the Rat Pituitary and Adrenal Glands
Authors: Montero-Hadjadje Maité , Pelletier Georges , Yon Laurent , Li Songyun , Guillemot Johann , Magoul Rabia , Tillet Yves , Vaudry Hubert , Anouar Youssef
Published: April 2011

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.
Distribution and Cellular Localization of Caldendrin Immunoreactivity in Adult Human Forebrain
Authors: Bernstein Hans-Gert , Seidenbecher Constanze I. , Smalla Karl-Heinz , Gundelfinger Eckart D. , Bogerts Bernhard , Kreutz Michael R.
Published: April 2011

We investigated by immunohistochemistry (IHC) the distribution of caldendrin, the founding member of a novel family of neuronal calcium-binding proteins closely related to calmodulin, in human forebrain. Caldendrin immunoreactivity was unevenly distributed, with prominent staining in the paleo- and neocortex, hippocampus, and hypothalamus. With the exception of the hypothalamus, labeling was restricted to the somatodendritic compartment of neurons. This distribution completely matches that reported in rat, indicating that the cellular function is most likely conserved among species. Therefore, one prerequisite for functional studies in rodent models aimed at elucidation of mechanisms with relevance for humans can be based on the present findings.
Concentration of Caveolin-3 at the Neuromuscular Junction in Young and Old Rat Skeletal Muscle Fibers
Authors: Carlson Bruce M. , Carlson Jean A. , Dedkov Eduard I. , McLennan Ian S.
Published: April 2011

Caveolin-3, a muscle-specific member of the caveolin family, is strongly localized to the neuromuscular junction (NMJ) in adult rat muscle fibers, where it co-localizes with α-bungarotoxin staining. In 24-month-old rats, less distinct staining corresponds with the normal aging changes in the NMJ. After denervation, the pattern and intensity of staining begin to break up as early as 3 days, and by 10 days little staining remains. The functional implications of this concentration of caveolin-3 at the NMJ remain obscure, but it is possible that its absence could account for some of the phenotypic characteristics of individuals with caveolin-3 mutations.
Double Autoimmunostaining with Glycine Treatment
Authors: Hasui Kazuhisa , Takatsuka Tomio , Sakamoto Ryoichi , Matsushita Sachie , Tsuyama Shin-ichiro , Izumo Shuji , Murata Fusayoshi
Published: April 2011

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase–hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or No-vaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.
Ultrastructural Analysis of DNA Complexes During Transfection and Intracellular Transport
Authors: Cartier Régis , Velinova Maria , Lehman Cathleen , Erdmann Bettina , Reszka Regina
Published: April 2011

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2–1-μm aggregates composed of 30–50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.
Leptin Expression in the Rat Ovary Depends on Estrous Cycle
Authors: Archanco Marta , Muruzábal Francisco J. , Llopiz Diana , Garayoa Mercedes , Gómez–Ambrosi Javier , Frühbeck Gema , Burrell María A.
Published: April 2011

Leptin is a hormone originally identified in adipocytes. It is involved in the regulation of fat deposition and energy expenditure and in other functions, such as reproduction. The presence of leptin has been reported in several reproductive organs. However, few studies have addressed its expression in the ovary. Moreover, the existing information is not consistent with regard to the particular cell types responsible for leptin expression. In this work we studied the distribution of leptin in the rat ovary by immunohistochemistry (IHC) and in situ hybridization (ISH). Leptin staining was found in steroid-producing cells: thecal, luteal, and interstitial cells. The strongest signal with both techniques was found in the cytoplasm of oocytes. A weak reaction for leptin mRNA was detected in granulosa of all growing follicles, although leptin protein was found only in the mature follicle. Western blotting analysis detects a strongly reactive 16-kD band, giving further support to the presence of leptin in the rat ovary. Variations in this immunoreactive band were found throughout the estrous cycle. Localization of leptin in the ovary may contribute to a better understanding of female reproductive function.
Apolipoprotein D Expression in Human Brain Reactive Astrocytes
Authors: del Valle Eva , Navarro Ana , Astudillo Aurora , Tolivia Jorge
Published: April 2011

Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipo-protein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.
Perlecan, the Multidomain Heparan Sulfate Proteoglycan of Basement Membranes, Is also a Prominent Component of the Cartilaginous Primordia in the Developing Human Fetal Spine
Authors: Melrose James , Smith Susan , Ghosh Peter , Whitelock John
Published: April 2011

The aim of this study was to localize perlecan in human fetal spine tissues. Human fetal spines (12–20 weeks; n=6) were fixed in either Histochoice or 10% neutral buffered formalin, routinely processed, paraffin-embedded, and 4-μm sagittal sections were cut and stained with toluidine blue, H&E, and von Kossa. Perlecan, types I, II, IV, and X collagen, CD-31, aggrecan core protein, and native and Δ-HS 4, 5 hexuronate stub epitopes were immunolocalized. Toluidine blue staining visualized the cartilaginous vertebral body (VB) rudiments and annular lamellae encompassing the nucleus pulposus (NP). Von Kossa staining identified the VB primary center of ossification. Immunolocalization of type IV collagen, CD-31, and perlecan delineated small blood vessels in the outer annulus fibrosus (AF) and large canals deep within the VBs. Perlecan and type X collagen were also prominently expressed by the hypertrophic vertebral growth plate chondrocytes. Aggrecan was extracellularly distributed in the intervertebral disk (IVD) with intense staining in the posterior AF. Notochordal tissue stained strongly for aggrecan but negatively for perlecan and types I and II collagen. Type I collagen was prominent in the outer AF and less abundant in the NP, while type II collagen was localized throughout the IVD and VB. The immunolocalization patterns observed indicated key roles for perlecan in vasculogenic, chondrogenic, and endochondral ossification processes associated with spinal development.
Follicular Expression of c-Kit/SCF and Inhibin-α in Mouse Ovary During Development
Authors: Kang Jae Seong , Lee Chang Joo , Lee Jong Min , Rha Joong Yeol , Song Kang Won , Park Moon Hyang
Published: April 2011

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-α at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-α was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-α was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-α is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.
Expression and Localization of Prohormone Convertase PC1 in the Calcitonin-producing Cells of the Bullfrog Ultimobranchial Gland
Authors: Yaoi Yuichi , Suzuki Masakazu , Tomura Hideaki , Kurabuchi Shingo , Sasayama Yuichi , Tanaka Shigeyasu
Published: April 2011

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.
Morphofunctional Studies of the Glomerular Wall in Mice Lacking Entactin-1
Authors: Lebel Simon-Philippe , Chen Yong , Gingras Diane , Chung Albert E. , Bendayan Moise
Published: April 2011

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the α3- and the αv-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of αv was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.
Expression of Myosin Heavy Chain Isoforms in Rat Soleus Muscle Spindles After 19 Days of Hypergravity
Authors: Picquet Florence , De-Doncker Laurent , Falempin Maurice
Published: April 2011

The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and α-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and α-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, α-cardiac, and slow-tonic.