bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental Behavior of Embryonic Myogenic Progenitors Transplanted into Adult Muscle as Revealed by Desmin LacZ Recombinant Gene
Authors: Auda-Boucher Gwenola , Rouaud Thierry , Fontaine-Pérus Josiane , Le Grand Fabien , Gardahaut Marie-France
Published: April 2011

We studied the behavior of myogenic progenitors from donor desmin+/– LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of β-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.
Chromosomal Variations Within Aneuploid Cancer Lines
Authors: Isaka Takahiro , Nestor Andrea L. , Takada Tadahiro , Allison David C.
Published: April 2011

Aneuploid cancers exhibit a wide spectrum of clinical aggressiveness, possibly because of varying chromosome compositions. To test this, karyotypes from the diploid CCD-34Lu fibroblast and the aneuploid A549 and SUIT-2 cancer lines underwent fluorescence in situ hybridization (FISH) and DAPI counterstaining. The number of DAPI-stained and FISH-identified chromosomes, 1–22, X,Y, as well as structural abnormalities, were counted and compared using the χ2, Mann–Whitney rank sum test and the Levene' equality of variance. Virtually all of the evaluable diploid CCD-34Lu karyotypes had 46 chromosomes with two normal-appearing homologues. The aneuploid chromosome numbers per karyotype were highly variable, averaging 62 and 72 for the A549 and SUIT-2 lines, respectively. However, the A549 chromosome numbers were more narrowly distributed than the SUIT-2 karyotype chromosome numbers. Furthermore, 25% of the A549 chromosomes had structural abnormalities compared to only 7% of the SUIT-2 chromosomes. The chromosomal compositions of the aneuploid A549 and SUIT-2 cancer lines are widely divergent, suggesting that diverse genetic alterations, rather than chance, may govern the chromosome makeups of aneuploid cancers.
Clusterin Expression in the Early Process of Pancreas Regeneration in the Pancreatectomized Rat
Authors: Min Bon-Hong , Kim Byeong-Moo , Lee Seung-Hee , Kang Sang-Wook , Bendayan Moise , Park In-Sun
Published: April 2011

We have previously reported upregulation of clusterin at the time of islet cell regeneration after β-cell injury. This led us to speculate that clusterin might be involved in the neogenic regeneration of the pancreas. Clusterin expression was examined throughout the process of pancreatic neogenesis in pancreatectomized rats. For in vitro analysis, duct cells were isolated from the rat pancreas and clusterin cDNA was transfected for its overexpression. Clusterin and its mRNA increased significantly in the early phase of regeneration, particularly at 1–3 days after pancreatectomy. Clusterin was transiently expressed in the differentiating acinar cells but faded afterwards. Interestingly, these clusterin cells were negative for PCNA (proliferating cell nuclear antigen), whereas most epithelial cells in ductules in the regenerating tissue showed extensive proliferative activity. Clusterin expression was also detected in some endocrine cells of the regenerating tissue. Transfection of clusterin cDNA into primary cultured duct cells resulted in a 2.5-fold increase in cell proliferation and induced transformation of non-differentiated duct cells into differentiated cells displaying cytokeratin immunoreactivity. Taken together, these results suggest that clusterin may play essential roles in the neogenic regeneration of pancreatic tissue by stimulating proliferation and differentiation of duct cells.
Localization and Identification of Schistosoma mansoni/KLH-crossreactive Components in Infected Mice
Authors: Thors Cecilia , Linder Ewert
Published: April 2011

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.
Expression and Localization of Vesicular Glutamate Transporters in Pancreatic Islets, Upper Gastrointestinal Tract, and Testis
Authors: Hayashi Mitsuko , Morimoto Riyo , Yamamoto Akitsugu , Moriyama Yoshinori
Published: April 2011

The wide-ranging expression of glutamate receptors in peripheral tissues suggests an unexpectedly wider role(s) of l-glutamate as an intercellular signaling molecule. However, the peripheral glutamatergic system is poorly understood, partly because the sites of l-glutamate signal appearance are less well characterized. Vesicular glutamate transporters (VGLUTs) are potential probes for the sites of vesicular storage and subsequent secretion of l-glutamate. In this study we raised specific polyclonal antibodies against two VGLUT isoforms, VGLUT1 and VGLUT2, and investigated their localization in peripheral tissues of rat. We detected the expression of either VGLUT1 or VGLUT2, or both, in pancreas, stomach, intestine, and testis. In pancreas, VGLUT1 and VGLUT2 are present in pancreatic polypeptide-containing secretory granules in F-cells in the islets of Langerhans. In stomach, VGLUT2 is abundant in the antrum and pylorus and is present in a subset of pancreatic polypeptide-containing cells. In intestine, VGLUT2 is abundant in the ileum and is co-localized with glucagon-like immunoreactive peptide and polypeptide YY (PYY). In testis, VGLUT2 is expressed and localized in the outer acrosomal membrane of spermatids, where KA1 and GluR5, kainate receptor subunits, are almost always localized. Taken together, these results strongly suggest the occurrence of a peripheral glutamatergic system in the gastroenteropancreatic system and testis.
Perlecan and Tumor Angiogenesis
Authors: Jiang Xinnong , Couchman John R.
Published: April 2011

Perlecan is a major heparan sulfate proteoglycan (HSPG) of basement membranes (BMs) and connective tissues. The core protein of perlecan is divided into five domains based on sequence homology to other known proteins. Commonly, the N-terminal domain I of mammalian perlecan is substituted with three HS chains that can bind a number of matrix molecules, cytokines, and growth factors. Perlecan is essential for metazoan life, as shown by genetic manipulations of nematodes, insects, and mice. There are also known human mutations that can be lethal. In vertebrates, new functions of perlecan emerged with the acquisition of a closed vascular system and skeletal connective tissues. Many of perlecan's functions may be related to the binding and presentation of growth factors to high-affinity tyrosine kinase (TK) receptors. Data are accumulating, as discussed here, that similar growth factor-mediated processes may have unwanted promoting effects on tumor cell proliferation and tumor angiogenesis. Understanding of these attributes at the molecular level may offer opportunities for therapeutic intervention.
Network Organization of Interstitial Connective Tissue Cells in the Human Endolymphatic Duct
Authors: Hultgård-Ekwall Anna-Karin H. , Couloigner Vincent , Rubin Kristofer , Rask-Andersen Helge
Published: April 2011

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.
Localization of Cholecystokinin Receptor Subtypes in the Endocine Pancreas
Authors: Morisset Jean , Julien Sophie , Lainé Jean
Published: April 2011

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCKA and CCKB receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCKA RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCKB RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCKB R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCKA and CCKB receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.
Vimentin-positive Cells in the Epithelium of Rabbit Ileal Villi Represent Cup Cells but not M-cells
Authors: Ramirez Carolina , Gebert Andreas
Published: April 2011

Membranous (M)-cells are specialized epithelial cells of the Peyer's patch domes that transport antigens from the intestinal lumen to the lymphoid tissue. Vimentin is a reliable marker for M-cells in rabbits. Using immunohistochemistry (IHC), a subpopulation of epithelial cells has recently been identified in ordinary rabbit ileal villi, which are vi-mentin-positive and share morphological characteristics with the M-cells of the domes. To test the hypothesis that these cells represent M-cells outside the organized lymphoid tissue, lectin labeling and tracer uptake experiments were performed. Lectins specific for N-acetyl-glucosamine oligomers selectively bound to the vimentin-positive villous cells but not to M-cells in the domes. Microbeads instilled into the ileal lumen were taken up by M-cells within 45 min but not by the vimentin-positive cells in the villi. Lectin-gold labeling on ultrathin sections revealed that the lectin binding sites were located in the brush border and in vesicles in the apical cytoplasm. The vimentin/lectin-positive cells shared ultrastructural characteristics with the so-called “cup cells.” We conclude (a) that the vimentin-positive cells in ordinary villi represent cup cells but not M-cells, (b) that they are readily detectable by (GlucNAc)N-specific lectins, and (c) that they do not transcytose experimental tracers. Although the specific function of cup cells is still obscure, they most probably represent a cell type distinct from M-cells of the domes with respect to both function and expression of the two new markers.
Actin Filament Organization of Foot Processes in Rat Podocytes
Authors: Ichimura Koichiro , Kurihara Hidetake , Sakai Tatsuo
Published: April 2011

The foot processes of podocytes possess abundant microfilaments and modulate glomerular filtration. We investigated the actin filament organization of foot processes in adult rat podocytes and the formation of the actin cytoskeletal system of immature podocytes during glomerulogenesis. Electron microscopy revealed two populations of actin cytoskeletons in foot processes of adult podocytes. One is the actin bundle running above the level of slit diaphragms and the other is the cortical actin network located beneath the plasmalemma. Immunogold labeling for actin-binding proteins demonstrated that oí-actinin and synaptopodin were localized in the actin bundle, whereas cortactin was in the cortical actin network. Immunofluorescence labeling for actin-binding proteins in immature podocyte showed that α-actinin was localized at the level of the junctional complex, whereas cortactin was distributed beneath the entire plasmalemma. Synaptopodin was first observed along the basal plasmalemma from the advanced S-shaped body to the capillary loop stage. We conclude that foot processes have specialized actin filamentous organization and that its establishment is associated with the expression and redistribution of actin-binding proteins during development.
Localization of a Brain Sulfotransferase, SULT4A1, in the Human and Rat Brain: An Immunohistochemical Study
Authors: Liyou Nancy E. , Buller Kathryn M. , Tresillian Michael J. , Elvin Christopher M. , Scott Heather L. , Dodd Peter R. , Tannenberg Anthony E.G. , McManus Michael E.
Published: April 2011

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.
Correlation of Staining for LKB1 and COX-2 in Hamartomatous Polyps and Carcinomas from Patients with Peutz–Jeghers Syndrome
Authors: Wei Chongjuan , Amos Christopher I. , Rashid Asif , Sabripour Mahyar , Nations Linda , McGarrity Thomas J. , Frazier Marsha L.
Published: April 2011

Germline mutations of the LKB1 gene lead to Peutz–Jeghers syndrome (PJS), which is associated with a predisposition to gastrointestinal polyposis and cancer. In this study we tested for germline mutations of LKB1 in 11 patients with PJS from nine families and analyzed the expression patterns of the LKB1 and cyclo-oxgenase-2 (COX-2) proteins in 28 Peutz–Jeghers polyps (PJPs) and five carcinomas from these patients by immunohistochemical (IHC) analysis. In eight of those families we identified seven different mutations, which consisted of two splice site mutations, two nonsense mutations, one small inframe deletion, one frame-shift mutation, and one silent mutation. Immunostaining revealed nuclear and cytoplasmic expression of LKB1 protein in 23 PJPs and five carcinomas, nuclear expression alone in one PJP, and loss of LKB1 protein expression in four PJPs, indicating a heterogeneous LKB1 expression pattern in PJPs. Overexpression of COX-2 was detected in 23 (82%) of 28 PJPs and in all carcinomas. Despite heterogeneity in staining of LKB1 among individuals and even among samples from the same individual, we found statistically significant correlations in staining of LKB1 relative to COX-2. These results suggest that COX-2 plays a role in tumorigenesis in PJS and may therefore be considered as a potential target for PJS chemoprevention.
Expression of Gastric Gland Mucous Cell-Type Mucin in Normal and Neoplastic Human Tissues
Authors: Nakajima Kosei , Ota Hiroyoshi , Zhang Mu Xia , Sano Kenji , Honda Takayuki , Ishii Keiko , Nakayama Jun
Published: April 2011

Gastric gland mucous cells produce class III mucin, which is also found in Brunner's glands and mucous glands along the pancreaticobiliary tract, and in metaplasia and adenocarcinomas differentiating towards gastric mucosa. Recently, we showed that class III mucin possesses GlcNAcα1→4Galβ→R, formed by α1,4- N-acetylglucosaminyltransferase (α4GnT). Examining the tissue-specific expression of mucin epitopes is useful to clarify cell-lineage differentiation and to identify the site of origin of metastatic carcinomas in histological specimens. Formalin-fixed, paraffin-embedded tissue sections from esophagus, stomach, colon, liver, pancreas, lung, kidney, prostate, breast, and salivary gland resected for carcinoma, as well as salivary gland adenoma, colon adenoma, and metastatic adenocarcinoma of lymph nodes from stomach, pancreas, colon, and breast, were immunostained for MUC6, α4GnT, and GlcNAcα1→4Galβ→R. These were all expressed in normal, metaplastic, and adenocarcinoma tissues of stomach, pancreas, and bile duct, and in pulmonary mucinous bronchioloalveolar carcinomas. Cells expressing α4GnT uniformly expressed GlcNAcα1→4Galβ→R. Only MUC6 was expressed in normal salivary glands, pancreas, seminal vesicles, renal tubules, and colon adenomas, and in normal tissue and adenocarcinomas of prostate and breast. No tissues showed immunoreactivity for α4GnT alone. Immunohistochemistry (IHC) profiles were similar for metastatic carcinomas and primary carcinoma tissues. The IHC profiles for MUC6, α4GnT, and GlcNAcα1→4Galβ→R may be diagnostically relevant.
Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates
Authors: Berlier Judith E. , Rothe Anca , Buller Gayle , Bradford Jolene , Gray Diane R. , Filanoski Brian J. , Telford William G. , Yue Stephen , Liu Jixiang , Cheung Ching-Ying , Chang Wesley , Hirsch James D. , Beechem Rosaria P. Haugland Joseph M. , Haugland Richard P.
Published: April 2011

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.
Immunohistochemical Assessment of the Peripheral Benzodiazepine Receptor in Human Tissues
Authors: Bribes Estelle , Carrière Dominique , Goubet Catherine , Galiègue Sylvaine , Casellas Pierre , Joêlle Simony-Lafontaine
Published: April 2011

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.
In Vivo Angiogenic Phenotype of Endothelial Cells and Pericytes Induced by Vascular Endothelial Growth Factor-A
Authors: Witmer Antonella N. , van Blijswijk Bart C. , van Noorden Cornelis J.F. , Vrensen Gijs F.J.M. , Schlingemann Reinier O.
Published: April 2011

VEGF-A is a major angiogenesis and permeability factor. Its cellular effects, which can be used as targets in anti-angiogenesis therapy, have mainly been studied in vitro using endothelial cell cultures. The purpose of the present study was to further characterize these effects in vivo in vascular endothelial cells and pericytes, in an experimental monkey model of VEGF-A-induced iris neovascularization. Two cynomolgus monkeys ( Macaca fascicularis) received four injections of 0.5 μg VEGF-A in the vitreous of one eye and PBS in the other eye. After sacrifice at day 9, eyes were enucleated and iris samples were snap-frozen for immunohistochemistry (IHC) and stained with a panel of antibodies recognizing endothelial and pericyte determinants related to angiogenesis and permeability. After VEGF-A treatment, the pre-existing iris vasculature showed increased permeability, hypertrophy, and activation, as demonstrated by increased staining of CD31, PAL-E, tPA, uPA, uPAR, Glut-1, and αvβ3 and αvβ5 integrins, VEGF receptors VEGFR-1, −2 and −3, and Tie-2 in endothelial cells, and of NG2 proteoglycan, uPA, uPAR, integrins and VEGFR-1 in pericytes. Vascular sprouts at the anterior surface of the iris were positive for the same antigens except for tPA, Glut-1, and Tie-2, which were notably absent. Moreover, in these sprouts VEGFR-2 and VEGFR-3 expression was very high in endothelial cells, whereas many pericytes were present that were positive for PDGFR-β, VEGFR-1, and NG2 proteoglycan and negative for α-SMA. In conclusion, proteins that play a role in angiogenesis are upregulated in both pre-existing and newly formed iris vasculature after treatment with VEGF-A. VEGF-A induces hypertrophy and loss of barrier function in pre-existing vessels, and induces angiogenic sprouting, characterized by marked expression of VEGFR-3 and lack of expression of tPA and Tie-2 in endothelial cells, and lack of α-SMA in pericytes. Our in vivo study indicates a role for α-SMA-negative pericytes in early stages of angiogenesis. Therefore, our findings shed new light on the temporal and spatial role of several proteins in the angiogenic cascade in vivo.
Individual Subtypes of Enteroendocrine Cells in the Mouse Small Intestine Exhibit Unique Patterns of Inositol 1,4,5-trisphosphate Receptor Expression
Authors: Wang Songyan , Liu Jianfeng , Li Lin , Wice Burton M.
Published: April 2011

Enteroendocrine cells are a complex population of intestinal epithelial cells whose hormones play critical roles in regulating gastrointestinal and whole-animal physiology. There are many subpopulations of enteroendocrine cells based on the major hormone(s) produced by individual cells. Intracellular calcium plays a critical role in regulating hormone release. Inositol 1,4,5-trisphophate (IP3) receptors regulate calcium mobilization from endoplasmic reticulum-derived calcium stores in many endocrine and excitatory cells and are expressed in the intestine. However, the specific subtypes of enteroendocrine cells that express these receptors have not been reported. Immunohistochemical (IHC) studies revealed that enteroendocrine cells did not express detectable levels of type 2 IP3 receptors, whereas nearly all enteroendocrine cells that produced chromogranin A and/or serotonin expressed type 1 and type 3 IP3 receptors. Conversely, enteroendocrine cells that produced glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, cholecystokinin, or somatostatin did not express detectable levels of any IP3 receptors. Subsets of enteroendocrine cells that produced substance P or secretin expressed type 1 (33% or 18%, respectively) and type 3 (10% or 62%, respectively) IP3 receptors. Thus, different subtypes of enteroendocrine cells, as well as individual cells that express a particular hormone, exhibit remarkable heterogeneity in the molecular machineries that regulate hormone release in vivo. These results suggest that therapeutic agents can be developed that could potentially inhibit or promote secretion of hormones from specific subtypes of enteroendocrine cells.
Correlated Endothelial Caveolin Overexpression and Increased Transcytosis in Experimental Diabetes
Authors: Pascariu Mirela , Bendayan Moïse , Ghitescu Lucian
Published: April 2011

We investigated the mechanism by which diabetes renders the capillary endothelium more permeable to macromolecules in the lungs of short-term diabetic rats. We used quantitative immunocytochemistry (ICC) to comparatively assess the permeability of alveolar capillaries to serum albumin in diabetic and normoglycemic animals. The effect of diabetes on the population of endothelial caveolae was evaluated by morphometry and by ICC and immunochemical quantification of the amount of caveolin in the whole cell or associated with the purified endothelial plasma membrane. A net increase in the amount of serum albumin taken up by the plasmalemmal vesicles of alveolar endothelial cells and transported to the interstitium was documented in diabetic animals. Interendothelial junctions were not permeated by albumin molecules. The alveolar endothelial cells of hyperglycemic rats contain more caveolae (1.3-fold), accounting for a larger (1.5-fold) fraction of the endothelial volume than those of normal animals. The hypertrophy of the caveolar compartment is accompanied by overexpression of endothelial caveolin 1. Although the aggregated thickness of the endothelial and alveolar epithelium basement membranes increases in diabetes (1.3-fold), the porosity of this structure appears to be unchanged. Capillary hyperpermeability to plasma macromolecules recorded in the early phase of diabetes is explained by an intensification of transendothelial vesicular transport and not by the destabilization of the interendothelial junctions.
Distribution of Substance P Receptor (Neurokinin-1 Receptor) in Normal Ovine Lung and During the Progression of Bronchopneumonia in Sheep
Authors: Grubor Branka , Ramirez-Romero Rafael , Gallup Jack M. , Bailey Ted B. , Ackermann Mark R.
Published: April 2011

Substance P contributes to the physiological homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. The distribution of substance P receptor [neurokinin-1 receptor (NK-1R)] in lungs of normal sheep and sheep with acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica was determined by immunohistochemistry (IHC). Three rabbit polyclonal antibodies generated to the same cytosolic C-terminal portion of NK-1R (residues 393-407) were tested. NK-1R immunoreactivity was traced in digital images and quantified with IPLAB software. There were no significant differences in NK-1R protein density between normal and infected lambs. Antibody 1 had the broadest distribution and intensity, and stained alveolar septae, smooth muscle cells of airways and vessels, epithelial cells of airways and alveoli, and submucosal glands. When all animals from the study were included, there was a trend towards decreased NK-1R immunoreactivity over time. The work suggests that (a) the density of NK-1R does not change during progression of bacterial ( M. haemolytica) bronchopneumonia, (b) NK-1R is widely distributed in ovine lung and decreases with age, and (c) antibodies to the same NK-1R cytosolic region can vary in specificity and affinity.
Phenotypic Changes in Dentition of Runx2 Homozygote-null Mutant Mice
Authors: Åberg Thomas , Cavender Adriana , Gaikwad Joel S. , Bronckers Antonius L.J.J. , Wang Xiuping , Waltimo-Sirén Janna , Thesleff Irma , D'Souza Rena N.
Published: April 2011

Genetic and molecular studies in humans and mice indicate that Runx2 (Cbfa1) is a critical transcriptional regulator of bone and tooth formation. Heterozygous mutations in Runx2 cause cleidocranial dysplasia (CCD), an inherited disorder in humans and mice characterized by skeletal defects, supernumerary teeth, and delayed eruption. Mice lacking the Runx2 gene die at birth and lack bone and tooth development. Our extended phenotypic studies of Runx2 mutants showed that developing teeth fail to advance beyond the bud stage and that mandibular molar organs were more severely affected than maxillary molar organs. Runx2 (−/−) tooth organs, when transplanted beneath the kidney capsules of nude mice, failed to progress in development. Tooth epithelial-mesenchymal recombinations using Runx2 (+/+) and (−/−) tissues indicate that the defect in mesenchyme cannot be rescued by normal dental epithelium. Finally, our molecular analyses showed differential effects of the absence of Runx2 on tooth extracellular matrix (ECM) gene expression. These data support the hypothesis that Runx2 is one of the key mesenchymal factors that influences tooth morphogenesis and the subsequent differentiation of ameloblasts and odontoblasts.
Temporal Changes in PTHrP, Bcl-2, Bax, Caspase, TGF-β, and FGF-2 Expression Following Growth Plate Irradiation With or Without Radioprotectant
Authors: Damron Timothy A. , Mathur Sharad , Horton Jason A. , Strauss Judith , Margulies Bryan , Grant William , Farnum Cornelia E. , Spadaro Joseph A.
Published: April 2011

This study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-β was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-β, Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.
Cocaine- and Amphetamine-regulated Transcript (CART) Is Expressed in Several Islet Cell Types During Rat Development
Authors: Wierup N. , Kuhar M. , Nilsson B.O. , Mulder H. , Ekblad E. , Sundler F.
Published: April 2011

Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic peptide widely expressed in the central and peripheral, including the enteric, nervous systems. CART is also expressed in pituitary endocrine cells, adrenomedullary cells, islet somatostatin cells, and in rat antral gastrin cells. We used immunocytochemistry (IHC) and in situ hybridization (ISH) to study CART expression in developing rat pancreas. We also examined co-expression of CART and islet hormones and developmental markers and the effect of CART on proliferation using clonal insulin cells (INS-1 832/13). A major portion of each of the islet cell types, except the ghrelin cells, expressed CART during a period before and around birth. Two weeks postnatally, CART expression was restricted to somatostatin cells. Pre- and early postnatally, many of the CART-expressing cells co-expressed cytokeratin 20 (CK20), a marker of duct cells and islet precursor cells, the trophic hormone gastrin, and a smaller subpopulation also harbored the proliferation marker Ki67. CART was also expressed in pancreatic nerve fibers, both sensory and autonomic, and in ganglion nerve cell bodies. Although highly expressed in the developing islets, CART did not affect proliferation of INS-1 cells. We have demonstrated that CART is expressed in several islet cell types during rat development but is restricted to somatostatin cells and neurons in the adult rat.
A Novel Technology Allowing Immunohistochemical Staining of a Tissue Section with 50 Different Antibodies in a Single Experiment
Authors: Furuya Tomoko , Ikemoto Kenzo , Kawauchi Shigeto , Oga Atsunori , Tsunoda Shin'ichi , Hirano Takashi , Sasaki Kohsuke
Published: April 2011

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a “multiplex-immunostain chip (MI chip).” The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.
Localization of Inhibins and Activins in Normal Endocrine Cells and Endocrine Tumors of the Gut and Pancreas: an Immunohistochemical and In Situ Hybridization Study
Authors: La Rosa Stefano , Uccella Silvia , Marchet Silvia , Capella Carlo , Lloyd Ricardo V.
Published: April 2011

Activins and inhibins, which belong to the TGFβ family, are composed of different combinations of α-, βA-, and βB-subunits, resulting in inhibin A (αβA), inhibin B (αβB), activin A (βAβA), activin B (βBβB), and activin AB (βAβB). They regulate several cell functions, acting as paracrine/autocrine factors. Their actions, which depend on binding to specific receptors, are also modulated by follistatin. Gastroenteropancreatic (GEP) endocrine cells and endocrine tumors (ETs) produce several growth factors, but it is not well known whether they express follistatin and the various inhibin/activin subunits. We studied their expression in 65 GEP ETs using immunohistochemistry (IHC) and in situ hybridization (ISH). The α-subunit and follistatin were not identified in normal GEP endocrine cells and were poorly expressed in ETs. A βA-subunit immunoreactivity (IR) was detected in A-, G-, EC-, and GIP-cells, while βB-chain IR was present only in D-cells. The mRNAs encoding for these molecules were poorly expressed in normal tissues. βA- and βB-subunits were identified in several ETs by both IHC and ISH: βA-subunit mainly in G-cell and A-cell ETs, and βB-subunit in D-cell, A-cell, and EC-cell ETs. Our results demonstrate a differential expression of activin/inhibin subunits among different types of GEP endocrine cells and related tumors, suggesting a role in modulation of biological functions of these normal and neoplastic endocrine cells.
Expression of Lipoxygenase During Organogenic Nodule Formation from Hop Internodes
Authors: Fortes Ana Margarida , José Coronado Maria , Testillano Pilar S. , Risueño Maria del Carmen , Pais Maria Salomé
Published: April 2011

Study of lipoxygenase expression (LOX; EC 1.13.11.12) during organogenic nodule formation in hop ( Humulus lupulus var. Nugget) showed that LOXs are developmentally regulated throughout the process, suggesting their involvement in the response of internodes to wounding, nodule formation, and plantlet regeneration from these nodules. LOX activity and lipid peroxides exhibited a huge increase during the first week of culture, which may indicate a role for LOX and LOX products in response to wounding in hop, as reported for other systems. Western blotting analysis showed a de novo synthesis of LOX isoenzymes in response to wounding and the detection of three different isoenzymes. Confocal analysis of LOX immunofluorescence revealed the presence of the enzyme in cortical cells of induced internodes and in prenodular cells, mostly appearing as cytoplasmic spots. Some of them were identified as lipid bodies by cytochemical and double immunofluorescence assays, suggesting the involvement of a lipid body LOX during nodule formation. Immunogold labeling detected LOX in peroxisomes, lipid bodies, and plastids of nodular cells. Quantification of the labeling density provided statistical significance for the localization of LOX (three different isoenzymes) in the three compartments, which suggested a possible involvement of LOX in metabolic functions of these organelles during organogenic nodule formation and plantlet regeneration.
Expression and Distribution of Somatostatin Receptor Subtypes in the Pancreatic Islets of Mice and Rats
Authors: Ludvigsen Eva , Olsson Richard , Stridsberg Mats , Janson Eva T. , Sandler Stellan
Published: April 2011

Somatostatin acts on specific membrane receptors (sst1–5) to inhibit exocrine and endocrine functions. The aim was to investigate the distribution of sst1–5 in pancreatic islet cells in normal mice and rats. Pancreatic samples from five adult C57BL/6 mice and Sprague-Dawley rats were stained with antibodies against sst1–5 and insulin, glucagon, somatostatin, or pancreatic polypeptide (PP). A quantitative analysis of the co-localization was performed. All ssts were expressed in the pancreatic islets and co-localized on islet cells to various extents. A majority of the β-cells expressed sst1–2 and sst5 in mouse islets, while ≤50% in the rat expressed sst1–5. The expression of sst1–5 on α-cells did not differ much among species, with sst2 and sst5 being highly expressed. About 70% of the 8-cells expressed sst1–4 in the rat pancreas, whereas 50% of the islet cells expressed sst1–5 in the mouse. Furthermore, 60% of the PP-cells expressed sst1–5 in the mouse, while the rat islets had lower values. Co-expression with the four major islet hormones varies among species and sst subtypes. These similarities and differences are interesting and need further evaluation to elucidate their physiological role in islets.
Immunohistochemical Analysis of Advanced Human Breast Carcinomas Reveals Downregulation of Protein Kinase Cα
Authors: Kerfoot Christopher , Huang Weidong , Rotenberg Susan A.
Published: April 2011

Forty-six advanced-stage human breast carcinoma specimens were evaluated by immunohistochemistry for PKCα expression and compared with 25 samples of normal adjacent breast tissue. For normal tissue, the median staining of ductal epithelia was of moderate intensity. No staining was observed for 67% of tumor specimens, and only 4% showed intensities greater than the median observed in normal tissue. Faint to moderate PKCα staining was observed in the stroma, inflammatory cells, and fibroblasts of tumors but was absent in normal tissue. These findings demonstrate that downregulation of PKCα protein occurs in epithelial cells of advanced breast tumors ( p<0.001).
Expression of Notch Receptors and Ligands in the Adult Gut
Authors: Sander Guy R. , Powell Barry C.
Published: April 2011

The Notch signaling pathway has become recognized as a vitally important pathway in regulating proliferative/differentiative decisions and cell fate. To explore the involvement of the Notch pathway in adult gut, we investigated the expression of Notch receptors and their ligands by Northern blotting and in situ hybridization. Notch receptors and ligands were expressed in both proliferative and post-mitotic cells throughout adult rat gut, variously in epithelial, immune, and endothelial cells. Expression of Notch1, Jagged1, and Jagged2 frequently overlapped, whereas Notch2 expression was restricted to specific crypt cells, the lamina propria of the large intestine, and Peyer's patch lymphocytes. We propose that the expression of multiple Notch receptors and ligands in a range of different intestinal cell types indicates that this signaling pathway underpins many of the processes involved in the maintenance and function of the adult gut.
Involvement of Myosin II in Dopamine-induced Reorganization of the Lactotroph Cell's Actin Cytoskeleton
Authors: Vitale María L. , Carbajal M. Eloísa
Published: April 2011

We have shown that dopamine (DA), an inhibitor of prolactin secretion from anterior pituitary lactotrophs, stabilizes the cortical actin cytoskeleton. DA-induced cortical actin stabilization is accompanied by cytoplasmic actin cable disassembly and cell rounding up. Our aim was to identify the mechanisms involved in DA-induced stabilization of the lactotroph's actin cytoskeleton. Here we show that DA increased the association of myosin II with the cell cortex, suggesting that DA facilitates actin-myosin interaction to stabilize cortical actin filaments. This notion was supported by the finding that inhibitors of actin-myosin interaction blocked DA-evoked morphological responses. In addition, our results showed that DA-induced myosin association with the cell periphery may be mediated by inhibition of Rac1/Cdc42-dependent pathways, whereas, DA-induced cytoplasmic actin filament disassembly may be mediated by the inhibition of MLCK- and RhoA-dependent pathways. In conclusion, the present results provide evidence that myosin II is involved in the DA-induced remodeling of actin filaments in lactotrophs, and that DA-induced cortical actin filament assembly and stabilization involve the translocation of myosin II to the cell cortex. This effect requires, among other things, inhibition of the Rac1/Cdc42-dependent signaling pathway.
Uptake and Rapid Transfer of Fluorescent Ceramide Analogues to Acidosomes (Late Endosomes) in Paramecium
Authors: Iwamoto Masaaki , Allen Richard D.
Published: April 2011

The ciliated protozoan Paramecium incorporates sphingolipids into its cell membranes. However, it is still unclear if these sphingolipids are metabolically synthesized in the cell or if their precursors are taken up from exogenous materials. Here we studied the route of uptake of fluorescence-labeled analogues of ceramide. Fluorescent ceramide was taken up rapidly independent of phagosome formation. Cold treatment caused a decrease in uptake, while reduction in the amount of cytosolic ATP induced by NaN3 and deoxyglucose resulted in accumulation without internalization of fluorescence at the plasma membrane. These results suggest that uptake of fluorescent ceramide occurs at the plasma membrane, that it is an ATP-dependent process, and that it is not a result of simple diffusion. At first intracellular fluorescence appeared principally in the posterior half of the cell and then spread throughout the cytosol. In particular, a high accumulation of fluorescence occurred in association with acidosomes (late endosome or multivesicular body-like vesicles) that bind to the surface of nascent and young phagosomes. Therefore, in the Paramecium cell a significant proportion of ceramide apparently enters the cell by endocytosis and is quickly relayed to acidosomes along the endocytic pathway before becoming part of the digestive vacuole (phagoacidosome) membrane.
Sorting of Furin in Polarized Epithelial and Endothelial Cells: Expression Beyond the Golgi Apparatus
Authors: Mayer Gaétan , Boileau Guy , Bendayan Moïse
Published: April 2011

The conversion of proteins into their mature forms underlies the functionality of many fundamental cellular pathways. One posttranslational modification leading to maturation of precursor proteins consists of the cleavage of their prodomain at pairs of basic amino acids by enzymes of the subtilisin-like mammalian proprotein convertase family. One of these enzymes, furin, acts in the constitutive secretory pathway of almost every cell type. However, in spite of furin's major roles in many pathophysiological processes, the exact subcellular sites of processing and activation of its substrates remain elusive. In this study, furin antigenic sites were tracked in subcellular compartments of various tissues and corresponding cell lines by high-resolution immunogold electron microscopy, Western blotting, cell transfection, and in vivo gene delivery of the furin cDNA. In addition to the Golgi apparatus, furin was assigned to endosomes and plasma membranes of polarized intestinal and renal epithelial cells and endothelial cells of the continuous, fenestrated, and discontinuous capillaries. Roles of furin in endothelial permeability, basement membrane turnover, and shedding of transmembrane proteins are supported by our data.