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Kazakov EP, Kireev II, Golyshev SA. Techniques for Selective Labeling of Molecules and Subcellular Structures for Cryo-Electron Tomography. BIOCHEMISTRY. BIOKHIMIIA 2025; 90:173-187. [PMID: 40254397 DOI: 10.1134/s0006297924604015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/07/2024] [Revised: 01/09/2025] [Accepted: 01/20/2025] [Indexed: 04/22/2025]
Abstract
Electron microscopy (EM) is one of the most efficient methods for studying the fine structure of cells with a resolution thousands of times higher than that of visible light microscopy. The most advanced implementation of electron microscopy in biology is EM tomography of samples stabilized by freezing without water crystallization (cryoET). By circumventing the drawbacks of chemical fixation and dehydration, this technique allows investigating cellular structures in three dimensions at the molecular level, down to resolving individual proteins and their subdomains. However, the problem of efficient identification and localization of objects of interest has not yet been solved, thus limiting the range of targets to easily recognizable or abundant subcellular components. Labeling techniques provide the only way for locating the subject of investigation in microscopic images. CryoET imposes conflicting demands on the labeling system, including the need to introduce into a living cell the particles composed of substances foreign to the cellular chemistry that have to bind to the molecule of interest without disrupting its vital functions and physiology of the cell. This review examines both established and prospective methods for selective labeling of proteins and subcellular structures aimed to enable their localization in cryoET images.
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Affiliation(s)
- Evgeny P Kazakov
- Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.
- Department of Cell Biology and Histology, Faculty of Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
| | - Igor I Kireev
- Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
- Department of Cell Biology and Histology, Faculty of Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
| | - Sergei A Golyshev
- Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
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Laboux O, Dion N, Arana-Chavez V, Ste-Marie LG, Nanci A. Microwave Irradiation of Ethanol-fixed Bone Improves Preservation, Reduces Processing Time, and Allows Both Light and Electron Microscopy on the Same Sample. J Histochem Cytochem 2016; 52:1267-75. [PMID: 15385573 DOI: 10.1177/002215540405201003] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Methylmethacrylate (MMA) embedding is routinely used for histomorphometry of undecalcified bone preserved by prolonged immersion in ethanol, a procedure that yields poor ultrastructural detail. Because microwave irradiation (MWI) facilitates penetration of fixatives, we have investigated whether it can improve preservation by ethanol. Rat tibiae, some labeled with tetracycline, and a human iliac crest biopsy were immersed in 70% ethanol and dehydrated, both under MWI, for a total processing time of ~7 hr. Controls were not irradiated, and all specimens were embedded in MMA at 4C. They were then processed for histomorphometry, histochemistry, structural analysis, and immunolabeling. The results showed that histological preservation was improved with MWI. Static bone formation and resorption parameters and rate of mineral apposition were similar to those of conventionally processed specimens. Mineral distribution, as visualized by von Kossa staining and backscattered electron imaging, was not affected. Alkaline phosphatase and tartrate-resistant acid phosphatase activity, as well as immunolocalization of bone sialoprotein and osteopontin, were readily visualized. Ultrastructurally, osteopontin exhibited a typical distribution in mineralization foci, between calcified collagen fibrils, and at cement lines. These data show that MWI improves preservation and permits application of a broad spectrum of analytical methodologies on the same bone sample while considerably reducing processing time.
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Affiliation(s)
- Olivier Laboux
- Department of Stomatology, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7.
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Nanci A, Wazen RM, Zalzal SF, Fortin M, Goldberg HA, Hunter GK, Ghitescu DL. A Tracer Study with Systemically and Locally Administered Dinitrophenylated Osteopontin. J Histochem Cytochem 2016; 52:1591-600. [PMID: 15557213 DOI: 10.1369/jhc.4a6452.2004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a “surgical window” in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected.
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Affiliation(s)
- Antonio Nanci
- Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7.
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Gingras D, Bendayan M. Evaluation of Pancreatic Amylase mRNA upon Cholinergic Stimulation of Secretion. J Histochem Cytochem 2016; 53:93-103. [PMID: 15637342 DOI: 10.1177/002215540505300111] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
The primary function of the exocrine pancreas consists of the synthesis and secretion of several digestive enzymes. It is well established that amylase secretion by rat pancreatic tissue or by isolated acinar cells in culture can be stimulated by the cholinergic agonist carbachol. However, the effect of this secretagogue on enzyme synthesis remains unclear. Some studies demonstrated increases in rates of synthesis, whereas others reported increases in secretion with or without decreases in synthesis. We have evaluated changes in pancreatic amylase mRNA and total RNA after a single injection of carbachol and under fasting conditions. Two approaches in molecular morphology were applied on rat pancreatic tissue: in situ hybridization and RNase A-gold. Both revealed decreases in RNA labeling at the level of the rough endoplasmic reticulum (RER) 5 min after stimulation of secretion and after fasting. Gradual recovery was registered 15 and 30 min after stimulation of secretion. Northern blotting confirmed drastic decreases in amylase mRNA 5 min after stimulation and after fasting. The combination of such different approaches has demonstrated drastic decreases in RNA at the RER level, reflecting declines in rates of synthesis at the translational level under all conditions tested.
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Affiliation(s)
- Diane Gingras
- Department of Pathology and Cell Biology, Université de Montréal, CP 6128 Succursale Centre-ville, Montréal, Québec, Canada H3T 1J4
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Poirier S, Hamouda HA, Villeneuve L, Demers A, Mayer G. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain. PLoS One 2016; 11:e0157230. [PMID: 27280970 PMCID: PMC4900664 DOI: 10.1371/journal.pone.0157230] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Accepted: 05/26/2016] [Indexed: 01/12/2023] Open
Abstract
PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.
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Affiliation(s)
- Steve Poirier
- Laboratory of Molecular Cell Biology, Montreal Heart Institute Research Center, QC, Canada
- Département de Pharmacologie, Faculté de Médecine, Université de Montréal, QC, Canada
| | - Hocine Ait Hamouda
- Laboratory of Molecular Cell Biology, Montreal Heart Institute Research Center, QC, Canada
- Département de Pharmacologie, Faculté de Médecine, Université de Montréal, QC, Canada
| | - Louis Villeneuve
- Laboratory of Molecular Cell Biology, Montreal Heart Institute Research Center, QC, Canada
| | - Annie Demers
- Laboratory of Molecular Cell Biology, Montreal Heart Institute Research Center, QC, Canada
| | - Gaétan Mayer
- Laboratory of Molecular Cell Biology, Montreal Heart Institute Research Center, QC, Canada
- Département de Pharmacologie, Faculté de Médecine, Université de Montréal, QC, Canada
- Faculté de Pharmacie, Université de Montréal, QC, Canada
- * E-mail:
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Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex. PLoS Biol 2016; 14:e1002365. [PMID: 26891179 PMCID: PMC4758718 DOI: 10.1371/journal.pbio.1002365] [Citation(s) in RCA: 85] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Accepted: 12/23/2015] [Indexed: 02/08/2023] Open
Abstract
The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes. Dissection of the nuclear pore complex—an ancient eukaryotic molecular machine—exposes a fundamental divergence in structure and function between yeast and humans versus trypanosomes and provides insights into the evolution of the nucleus. Much of the core architecture of the eukaryotic cell was established over one billion years ago. Significantly, many cellular systems possess lineage-specific features, and architectural and compositional variation of complexes and pathways that are likely keyed to specific functional adaptations. The nuclear pore complex (NPC) contributes to many processes, including nucleocytoplasmic transport, interactions with the nuclear lamina, and mRNA processing. We exploited trypanosome parasites to investigate NPC evolution and conservation at the level of protein–protein interactions and composition. We unambiguously assigned NPC components to specific substructures and found that the NPC structural scaffold is generally conserved, albeit with lineage-specific elements. However, there is significant variation in pore membrane proteins and an absence of critical components involved in mRNA export in fungi and animals (opisthokonts). This is reflected by the completely symmetric localization of all trypanosome nucleoporins, with the exception of the nuclear basket. This architecture is highly distinct from opisthokonts. We also identify features that suggest a Ran-dependent system for mRNA export in trypanosomes, a system that may presage distinct mechanisms of protein and mRNA transport in animals and fungi. Our study highlights that shared composition of macromolecular assemblies does not necessarily equate to shared architecture. Identification of lineage-specific features within the trypanosome NPC significantly advances our understanding of mechanisms of nuclear transport, gene expression, and evolution of the nucleus.
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Abstract
Low voltage transmission electron microscopy (LVTEM) was employed to examine biological tissues with accelerating voltages as low as 5kV. Tissue preparation was modified to take advantage of the low-voltage techniques. Treatments with heavy metals, such as post-fixation with osmium tetroxide, on block and counterstaining were omitted. Sections (40nm) were thinner than usual and generated highly contrasted images. General appearance of the cells remains similar to that of conventional TEM. New features were however revealed. The matrix of the pancreatic granules displays heterogeneity with partitions that may correspond to the inner-segregation of their secretory proteins. Mitochondria revealed the presence of the ATP synthase granules along their cristea. The nuclear dense chromatin displayed a honeycomb organization while distinct beads, nucleosomes, aligned along thin threads were seen in the dispersed chromatin. Nuclear pore protein complexes revealed their globular nature. The intercalated disks in cardiac muscle displayed their fine structural organization. These features correlate well with data described or predicted by cell and molecular biology. These new aspects are not revealed when thicker and conventionally osmicated tissue sections were examined by LVTEM, indicating that major masking effects are associated with standard TEM techniques. Immunogold was adapted to LVTEM further enhancing its potential in cell biology.
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Miron RJ, Bosshardt DD, Buser D, Zhang Y, Tugulu S, Gemperli A, Dard M, Caluseru OM, Chandad F, Sculean A. Comparison of the Capacity of Enamel Matrix Derivative Gel and Enamel Matrix Derivative in Liquid Formulation to Adsorb to Bone Grafting Materials. J Periodontol 2015; 86:578-87. [DOI: 10.1902/jop.2015.140538] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Chen L, Jacquet R, Lowder E, Landis WJ. Refinement of collagen-mineral interaction: a possible role for osteocalcin in apatite crystal nucleation, growth and development. Bone 2015; 71:7-16. [PMID: 25284158 DOI: 10.1016/j.bone.2014.09.021] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2014] [Revised: 09/25/2014] [Accepted: 09/26/2014] [Indexed: 11/23/2022]
Abstract
Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen. These proteins, as possibly related to mineral deposition, have not been definitively localized in vivo. The present study has reexamined their localization in the leg tendons of avian turkeys, a representative model of vertebrate mineralization. Immunocytochemistry of osteocalcin demonstrates its presence at the surface of, outside and within type I collagen while that of bone sialoprotein appears to be localized at the surface of or outside type I collagen. The association between osteocalcin and type I collagen structure is revealed optimally when calcium ions are added to the antibody solution in the methodology. In this manner, osteocalcin is found specifically located along the a4-1, b1, c2 and d bands defining in part the hole and overlap zones within type I collagen. From these data, while type I collagen itself may be considered a stereochemical guide for intrafibrillar mineral nucleation and subsequent deposition, osteocalcin bound to type I collagen may also possibly mediate nucleation, growth and development of platelet-shaped apatite crystals. Bone sialoprotein and osteocalcin as well, each immunolocalized at the surface of or outside type I collagen, may affect mineral deposition in these portions of the avian tendon.
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Affiliation(s)
- Ling Chen
- Department of Polymer Science, University of Akron, Akron, OH, USA
| | - Robin Jacquet
- Department of Polymer Science, University of Akron, Akron, OH, USA
| | - Elizabeth Lowder
- Department of Polymer Science, University of Akron, Akron, OH, USA
| | - William J Landis
- Department of Polymer Science, University of Akron, Akron, OH, USA.
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Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces. Cell Tissue Res 2014; 358:843-55. [PMID: 25193156 DOI: 10.1007/s00441-014-1989-3] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 08/07/2014] [Indexed: 10/24/2022]
Abstract
Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces.
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Bendayan M, Paransky E. Perspectives on low voltage transmission electron microscopy as applied to cell biology. Microsc Res Tech 2014; 77:999-1004. [PMID: 25164611 DOI: 10.1002/jemt.22428] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Revised: 08/06/2014] [Accepted: 08/06/2014] [Indexed: 11/06/2022]
Abstract
Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey-comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin-A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology.
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Affiliation(s)
- Moise Bendayan
- Department of Pathology and Cell Biology, University of Montreal (MB), Montreal, Quebec, Canada; Delong America Inc. (EP), Montreal, Quebec, Canada
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12
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Schlienger S, Campbell S, Claing A. ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion. Mol Biol Cell 2013; 25:17-29. [PMID: 24196838 PMCID: PMC3873888 DOI: 10.1091/mbc.e13-06-0335] [Citation(s) in RCA: 102] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The small GTPase ARF1 is overexpressed in invasive breast cancer cells. This ARF isoform controls MMP-9 activity to degrade the extracellular matrix by regulating invadopodia maturation and microvesicle shedding. The molecular mechanisms by which ARF1 controls invasiveness involve regulation of the Rho/MLC pathway. Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.
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Affiliation(s)
- Sabrina Schlienger
- Department of Pharmacology and Membrane Protein Research Group (GEPROM), Faculty of Medicine, Université de Montréal, Montréal, QC H3C 3J7, Canada
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Seehuber A, Dahint R. Conformation and Activity of Glucose Oxidase on Homogeneously Coated and Nanostructured Surfaces. J Phys Chem B 2013; 117:6980-9. [DOI: 10.1021/jp401906h] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Affiliation(s)
- A. Seehuber
- Applied Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg,
Germany
| | - R. Dahint
- Applied Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg,
Germany
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Miron RJ, D. Bosshardt D, Hedbom E, Zhang Y, Haenni B, Buser D, Sculean A. Adsorption of Enamel Matrix Proteins to a Bovine-Derived Bone Grafting Material and Its Regulation of Cell Adhesion, Proliferation, and Differentiation. J Periodontol 2012; 83:936-47. [DOI: 10.1902/jop.2011.110480] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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Odontogenic ameloblast-associated and amelotin are novel basal lamina components. Histochem Cell Biol 2012; 137:329-38. [PMID: 22231912 DOI: 10.1007/s00418-011-0901-4] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/08/2011] [Indexed: 10/14/2022]
Abstract
Odontogenic ameloblast-associated (ODAM) and amelotin (AMTN) are secreted by maturation stage ameloblasts and accumulate at the interface with enamel where an atypical basal lamina (BL) is present. This study aimed at determining and quantifying the ultrastructural distribution of ODAM and AMTN at the cell-tooth interface. Ultrathin sections of enamel organs from the early to mid- and late maturation stage of amelogenesis were processed for immunogold labeling with antibodies against ODAM, AMTN or with the lectins wheat germ agglutinin, Helix pomatia agglutinin (HPA) and Ricinus communis I agglutinin. Immunolabeling showed that both ODAM and AMTN localized to the BL. Quantitative analyses indicated that at the beginning of maturation there is a concentration of ODAM on the cell side of the BL while AMTN appears more concentrated on the enamel side. In the late maturation stage, such differential distribution is no longer apparent. All three lectins are bound to the BL. Competitive incubation with native lectins did not affect the binding efficiency of ODAM; however, AMTN binding was significantly reduced after incubation with HPA. In conclusion, ODAM and AMTN are bona fide components of the BL associated with maturation stage ameloblasts and they organize into different subdomains during the early maturation stage. The data also suggest that the BL is a dynamic structure that rearranges its organization as enamel maturation advances. Finally, the abrogation of AMTN antibody labeling by HPA supports the presence of O-linked sugars in the molecule and/or its close association with other O-glycosylated molecules.
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Scherzer P, Katalan S, Got G, Pizov G, Londono I, Gal-Moscovici A, Popovtzer MM, Ziv E, Bendayan M. Psammomys obesus, a particularly important animal model for the study of the human diabetic nephropathy. Anat Cell Biol 2011; 44:176-85. [PMID: 22025969 PMCID: PMC3195821 DOI: 10.5115/acb.2011.44.3.176] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2011] [Revised: 09/12/2011] [Accepted: 09/14/2011] [Indexed: 01/11/2023] Open
Abstract
The Psammomys obesus lives in natural desert habitat on low energy (LE) diet, however when maintained in laboratory conditions with high energy (HE) diet it exhibits pathological metabolic changes resembling those of type 2 diabetes. We have evaluated and correlated the histopathology, metabolic and functional renal alterations occurring in the diabetic Psammomys. Renal function determined by measuring glomerular filtration rate (GFR), protein excretion, protein/creatinine ratio and morpho-immunocytochemical evaluations were performed on HE diet diabetic animals and compared to LE diet control animals. The diabetic animals present a 54% increase in GFR after one month of hyperglycemic condition and a decrease of 47% from baseline values after 4 months. Protein excretion in diabetic animals was 5 folds increased after 4 months. Light microscopy showed an increase in glomeruli size in the diabetic Psammomys, and electron microscopy and immunocytochemical quantitative evaluations revealed accumulation of basement membrane material as well as frequent splitting of the glomerular basement membrane. In addition, glycogen-filled Armanni-Ebstein clear cells were found in the distal tubules including the thick ascending limbs of the diabetic animals. These renal complications in the Psammomys, including changes in GFR with massive proteinuria sustained by physiological and histopathological changes, are very similar to the diabetic nephropathy in human. The Psamommys obesus represents therefore a reliable animal model of diabetic nephropathy.
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Affiliation(s)
- Pnina Scherzer
- Nephrology and Hypertension Unit, Hadassah University Hospital, Jerusalem, Israel
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Bendayan M, Londono I, Paransky E. Compartmentalization of pancreatic secretory zymogen granules as revealed by low-voltage transmission electron microscopy. J Histochem Cytochem 2011; 59:899-907. [PMID: 21832147 DOI: 10.1369/0022155411418507] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.
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Affiliation(s)
- Moise Bendayan
- Department of Pathology and Cell Biology and Montreal Diabetes Center, University of Montreal, Canada.
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Trubiani O, Zalzal SF, Paganelli R, Marchisio M, Giancola R, Pizzicannella J, Bühring HJ, Piattelli M, Caputi S, Nanci A. Expression profile of the embryonic markers nanog, OCT-4, SSEA-1, SSEA-4, and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells. J Cell Physiol 2010; 225:123-31. [PMID: 20458727 DOI: 10.1002/jcp.22203] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Mesenchymal stem cells (MSCs) are self-renewing cells with the ability to differentiate into various mesodermal-derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL-MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL-MSCs contain a subpopulation of frizzled-9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA-1, and SSEA-4. They are additionally positive for nanog and Oct-4; two critical transcription factors directing self-renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP-10. The presence of nanog, Oct-4, SSEA-1, and SSEA-4 suggests that PDL-MSCs are less differentiated than bone marrow-derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled-9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells.
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Affiliation(s)
- Oriana Trubiani
- Department of Oral Science, University of Chieti-Pescara, Chieti, Italy.
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Cammisotto PG, Levy E, Bukowiecki LJ, Bendayan M. Cross-talk between adipose and gastric leptins for the control of food intake and energy metabolism. ACTA ACUST UNITED AC 2010; 45:143-200. [PMID: 20621336 DOI: 10.1016/j.proghi.2010.06.001] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/06/2010] [Indexed: 12/25/2022]
Abstract
The understanding of the regulation of food intake has become increasingly complex. More than 20 hormones, both orexigenic and anorexigenic, have been identified. After crossing the blood-brain barrier, they reach their main site of action located in several hypothalamic areas and interact to balance satiety and hunger. One of the most significant advances in this matter has been the discovery of leptin. This hormone plays fundamental roles in the control of appetite and in regulating energy expenditure. In accordance with the lipostatic theory stated by Kennedy in 1953, leptin was originally discovered in white adipose tissue. Its expression by other tissues was later established. Among them, the gastric mucosa has been shown to secrete large amounts of leptin. Both the adipose and the gastric tissues share similar characteristics in the synthesis and storage of leptin in granules, in the formation of a complex with the soluble receptor and a secretion modulated by hormones and energy substrates. However while adipose tissue secretes leptin in a slow constitutive endocrine way, the gastric mucosa releases leptin in a rapid regulated exocrine fashion into the gastric juice. Exocrine-secreted leptin survives the extreme hydrolytic conditions of the gastric juice and reach the duodenal lumen in an intact active form. Scrutiny into transport mechanisms revealed that a significant amount of the exocrine leptin crosses the intestinal wall by active transcytosis. Leptin receptors, expressed on the luminal and basal membrane of intestinal epithelial cells, are involved in the control of nutrient absorption by enterocytes, mucus secretion by goblet cells and motility, among other processes, and this control is indeed different depending upon luminal or basal stimulus. Gastric leptin after transcytosis reaches the central nervous system, to control food intake. Studies using the Caco-2, the human intestinal cell line, in vitro allowed analysis of the mechanisms of leptin actions on the intestinal mucosa, identification of the mechanisms of leptin transcytosis and understanding the modulation of leptin receptors by nutrients and hormones. Exocrine-secreted gastric leptin thus participates in a physiological axis independent in terms of time and regulation from that of adipose tissue to rapidly control food intake and nutrient absorption. Adipocytes and gastric epithelial cells are two cell types the metabolism of which is closely linked to food intake and energy storage. The coordinated secretion of adipose and gastric leptins ensures proper management of food processing and energy storage.
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Affiliation(s)
- Philippe G Cammisotto
- Department of Pathology and Cell Biology, University of Montreal, 2900 Boulevard Edouard-Montpetit, Montreal, QC, Canada.
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Receptor-Mediated Transcytosis of Leptin through Human Intestinal Cells In Vitro. Int J Cell Biol 2010; 2010:928169. [PMID: 20454702 PMCID: PMC2862316 DOI: 10.1155/2010/928169] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2009] [Accepted: 02/11/2010] [Indexed: 01/08/2023] Open
Abstract
Gastric Leptin is absorbed by duodenal enterocytes and released on the basolateral side towards the bloodstream. We investigated in vitro some of the mechanisms of this transport. Caco-2/15 cells internalize leptin from the apical medium and release it through transcytosis in the basal medium in a time- temperature-dependent and saturable fashion. Leptin receptors are revealed on the apical brush-border membrane of the Caco-2 cells. RNA-mediated silencing of the receptor led to decreases in the uptake and basolateral release. Leptin in the basal medium was found bound to the soluble form of its receptor. An inhibitor of clathrin-dependent endocytosis (chlorpromazine) decreased leptin uptake. Confocal immunocytochemistry and the use of brefeldin A and okadaic acid revealed the passage of leptin through the Golgi apparatus. We propose that leptin transcytosis by intestinal cells depends on its receptor, on clathrin-coated vesicles and transits through the Golgi apparatus.
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21
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Bendayan M. A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology. Biotech Histochem 2010. [DOI: 10.1080/10520290009068433] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
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Bendayan M, Londono I, Kemp BE, Hardie GD, Ruderman N, Prentki M. Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy. J Histochem Cytochem 2009; 57:963-71. [PMID: 19581628 DOI: 10.1369/jhc.2009.954016] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
Abstract
Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK alpha and beta subunits were located both in the cytosol and in close association with rosettes of glycogen particles (alpha particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the alpha(1) and alpha(2) subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for beta(1) was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the beta(1) subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.
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Affiliation(s)
- Moise Bendayan
- Department of Pathology and Cell Biology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada.
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Levy E, Ménard D, Delvin E, Montoudis A, Beaulieu JF, Mailhot G, Dubé N, Sinnett D, Seidman E, Bendayan M. Localization, function and regulation of the two intestinal fatty acid-binding protein types. Histochem Cell Biol 2009; 132:351-67. [PMID: 19499240 DOI: 10.1007/s00418-009-0608-y] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/13/2009] [Indexed: 01/20/2023]
Abstract
Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus. Immunoelectron microscopy revealed more intense labeling of L-FABP compared with I-FABP, accompanied with a heterogeneous distribution in the cytoplasm, microvilli and basolateral membranes. By western blot analysis, I- and L-FABP at 15 weeks of gestation appeared predominant in jejunum compared with duodenum, ileum, proximal and distal colon. Exploration of the maturation aspect documented a rise in L-FABP in adult tissues. Permanent transfections of Caco-2 cells with I-FABP cDNA resulted in decreased lipid export, apolipoprotein (apo) biogenesis and chylomicron secretion. Additionally, supplementation of Caco-2 with insulin, hydrocortisone and epidermal growth factor differentially modulated the expression of I- and L-FABP, apo B-48 and microsomal triglyceride transfer protein (MTP), emphasizing that these key proteins do not exhibit a parallel modulation. Overall, our findings indicate that the two FABPs display differences in localization, regulation and developmental pattern.
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Affiliation(s)
- Emile Levy
- Department of Nutrition, CHU-Sainte-Justine, University of Montreal, 3175 Côte Ste-Catherine Road, Montreal, QC, H3T 1C5, Canada.
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Zastre JA, Chan GNY, Ronaldson PT, Ramaswamy M, Couraud PO, Romero IA, Weksler B, Bendayan M, Bendayan R. Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line. J Neurosci Res 2009; 87:1023-36. [PMID: 18855943 DOI: 10.1002/jnr.21898] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
A major concern regarding the chronic administration of antiretroviral drugs is the potential for induction of drug efflux transporter expression (i.e., P-glycoprotein, P-gp) at tissue sites that can significantly affect drug distribution and treatment efficacy. Previous data have shown that the inductive effect of human immunodeficiency virus protease inhibitors (PIs) is mediated through the human orphan nuclear receptor, steroid xenobiotic receptor (SXR or hPXR). The objectives of this study were to investigate transport and inductive properties on efflux drug transporters of two PIs, atazanavir and ritonavir, at the blood-brain barrier by using a human brain microvessel endothelial cell line, hCMEC/D3. Transport properties of PIs by the drug efflux transporters P-gp and multidrug resistance protein 1 (MRP1) were assessed by measuring the cellular uptake of (3)H-atazanavir or (3)H-ritonavir in P-gp and MRP1 overexpressing cells as well as hCMEC/D3. Whereas the P-gp inhibitor, PSC833, increased atazanavir and ritonavir accumulation in hCMEC/D3 cells by 2-fold, the MRP inhibitor MK571 had no effect. P-gp, MRP1, and hPXR expression and localization were examined by Western blot analysis and immunogold cytochemistry at the electron microscope level. Treatment of hCMEC/D3 cells for 72 hr with rifampin or SR12813 (two well-established hPXR ligands) or PIs (atazanavir or ritonavir) resulted in an increase in P-gp expression by 1.8-, 6-, and 2-fold, respectively, with no effect observed for MRP1 expression. In hCMEC/D3 cells, cellular accumulation of these PIs appears to be primarily limited by P-gp efflux activity. Long-term exposure of atazanavir or ritonavir to brain microvessel endothelium may result in further limitations in brain drug permeability as a result of the up-regulation of P-gp expression and function.
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Affiliation(s)
- Jason A Zastre
- Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Ontario, Canada
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Ravid Z, Bendayan M, Delvin E, Sane AT, Elchebly M, Lafond J, Lambert M, Mailhot G, Levy E. Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors. Am J Physiol Gastrointest Liver Physiol 2008; 295:G873-85. [PMID: 18772361 DOI: 10.1152/ajpgi.90376.2008] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [(14)C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose (P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 (P < 0.02) and concomitantly decreased SR-BI protein mass (P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased (P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-alpha, LXR-beta, PPAR-beta, and PPAR-gamma along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.
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Affiliation(s)
- Z Ravid
- Research Centre, CHU-Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5
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26
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Cammisotto PG, Bendayan M. Adiponectin stimulates phosphorylation of AMP-activated protein kinase alpha in renal glomeruli. J Mol Histol 2008; 39:579-84. [PMID: 18941912 DOI: 10.1007/s10735-008-9198-6] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2008] [Accepted: 10/03/2008] [Indexed: 10/21/2022]
Abstract
Adiponectin receptor ADIPOR1 activates the intracellular second messenger AMP-activated protein kinase (AMPK) that participates in the control of the oxidative stress and apoptosis. This study reveals the presence of a functional ADIPOR1 receptor in all the cells of the renal glomeruli. Isolated glomeruli were incubated in vitro with adiponectin and proteins analysed by western blot. Electron microscopy using immunogold labeling was carried out on kidney sections. ADIPOR1 and catalytic AMPK sub-units alpha1 and alpha2 were revealed in normal rat glomeruli and incubation of freshly isolated rat glomeruli with either adiponectin or AICAR led to the activation by phosphorylation of catalytic AMPK. Electron microscopy localized with high resolution these proteins at the plasma membrane of the three glomerular cells, namely the endothelial, the mesangial and the podocyte cells, as well as on Bowman's capsule epithelial cells. It is concluded that glomerular cells express a functional adiponectin receptor ADIPOR1 which, through activation of AMPK, may play important roles in the control of oxidative stress and cell survival within the glomerulus.
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Affiliation(s)
- Philippe G Cammisotto
- Department of Pathology and Cell Biology, University of Montreal, 2900, bd Edouard-Montpetit, Montreal, QC, Canada, H3T 1J4
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Bendayan M, Gingras D, Ziv E, Haviv YS. Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells. Microsc Res Tech 2008; 71:659-62. [DOI: 10.1002/jemt.20603] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Abstract
OBJECTIVES Viral vector uptake into the pancreas is rare. The few viral vectors reported to transduce in vivo pancreatic islets after systemic injection required additional physical measures, such as direct pancreatic injection or hepatic vessel clamping. Because pancreatic islet uptake of the human polyomavirus family member BK virus was previously reported in hamsters after systemic administration, we hypothesized that SV40, a polyomavirus member remarkably similar to BK virus, may also infect the pancreas. METHODS We injected intravenously a low dose of SV40, unaided by any other physical or chemical means, and evaluated viral uptake by pancreatic islets and pancreatic exocrine tissue via polymerase chain reaction, Western blot, electron microscopy, immunofluorescent microscopy, and protein A-gold immunocytochemistry. RESULTS Pancreatic uptake of SV40 was comparable to other major organs (ie, liver and spleen). SV40 viral particles were detected in both pancreatic islets and acini. In pancreatic islets, all islet cell types were infected by SV40, albeit the infection rate of glucagon-producing alpha cells surpassed beta- and delta-islet cells. Low-dose SV40 administration was not sufficient to induce heterologous gene expression in the pancreas. CONCLUSIONS Our study shows that pancreatic islet and acinar cell uptake of SV40 is feasible with a single, low-dose intravenous injection. However, this dose did not result in gene delivery into the murine pancreas.
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Acevedo LM, Londono I, Oubaha M, Ghitescu L, Bendayan M. Glomerular CD34 expression in short- and long-term diabetes. J Histochem Cytochem 2008; 56:605-14. [PMID: 18319274 DOI: 10.1369/jhc.7a7354.2008] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Aging and diabetes are associated with exacerbated expression of adhesion molecules. Given their importance in endothelial dysfunction and their possible involvement in the alteration of glomerular permeability occurring in diabetes, we have evaluated expression of the sialomucin-type adhesion molecule CD34 in renal glomerular cells of normal and diabetic animals at two different ages by colloidal gold immunocytochemistry and immunoblotting. CD34 labeling was mostly assigned to the plasma membranes of glomerular endothelium and mesangial processes. Podocyte membranes were also labeled, but to a lesser degree. Short- and long-term diabetes triggers a substantial increase in immunogold labeling for CD34 in renal tissues compared with young normoglycemic animals. However, the level of labeling in old diabetic and healthy control rats is similar, suggesting that the effect of diabetes and aging on CD34 expression is similar but not synergistic. Western blotting of isolated glomerular fractions corroborated immunocytochemical results. Increased expression of CD34 may reflect its involvement in the pathogenesis of glomerular alterations related to age and diabetes. Alterations present in early diabetes, resembling those occurring with age, strengthen the concept that diabetes is an accelerated form of aging.
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Affiliation(s)
- Luz Marina Acevedo
- Department of Pathology and Cell Biology, Université de Montréal, Montréal QC H3T 1J4, Canada
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31
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Levy E, Trudel K, Bendayan M, Seidman E, Delvin E, Elchebly M, Lavoie JC, Precourt LP, Amre D, Sinnett D. Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats. Am J Physiol Gastrointest Liver Physiol 2007; 293:G1252-61. [PMID: 17916643 DOI: 10.1152/ajpgi.00369.2007] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.
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Affiliation(s)
- Emile Levy
- Department of Nutrition, Université de Montréal, Research Centre, CHU Sainte Justine, Montréal, Québec, Canada.
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Cammisotto PG, Gingras D, Bendayan M. Transcytosis of gastric leptin through the rat duodenal mucosa. Am J Physiol Gastrointest Liver Physiol 2007; 293:G773-9. [PMID: 17673543 DOI: 10.1152/ajpgi.00260.2007] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation. In another set of experiments, recombinant leptin was inserted in vivo into the duodenal lumen. Plasma levels of leptin were assessed by enzyme immunoassay and Western blot, and duodenal tissue was processed for immunocytochemistry. We first observed that leptin was found inside duodenal enterocytes from fed rats but not inside those from fasted ones. Stimulation of gastric secretion by a cholinergic agent led to rapid increases in plasma leptin levels (202 +/- 39%) except when the pylorus was clamped. Insertion of recombinant leptin into the duodenal lumen raised plasma leptin concentrations (558 +/- 34%) quite rapidly, whereas carrier solution without leptin had no effect. The use of FITC-tagged leptin reinforced these results. Light and electron microscopy revealed the cellular compartments involved in its transcytosis, namely, the enterocyte microvilli, the endocytotic vesicles, the Golgi complex, and the basolateral interdigitations. Leptin was also present in the lamina propria, in capillary endothelial cell plasmalemmal vesicles, and in capillary lumina. These results demonstrate that gastric exocrine leptin is internalized by duodenal enterocytes and delivered to the lamina propria and blood circulation. It may thus be able to play important paracrine and endocrine functions for the control of gastric emptying and nutrient absorption.
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Affiliation(s)
- Philippe G Cammisotto
- Department of Pathology and Cell Biology, University of Montreal, Montréal, Québec, Canada
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Babakhanian K, Bendayan M, Bendayan R. Localization of P-glycoprotein at the nuclear envelope of rat brain cells. Biochem Biophys Res Commun 2007; 361:301-6. [PMID: 17651695 DOI: 10.1016/j.bbrc.2007.06.176] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2007] [Accepted: 06/22/2007] [Indexed: 11/26/2022]
Abstract
P-glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.
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Affiliation(s)
- Karlo Babakhanian
- Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ont., Canada M5S 3M2
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Emelyanov VV, Vyssokikh MY. On the nature of obligate intracellular symbiosis of rickettsiae--Rickettsia prowazekii cells import mitochondrial porin. BIOCHEMISTRY (MOSCOW) 2006; 71:730-5. [PMID: 16903827 DOI: 10.1134/s0006297906070054] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.
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Affiliation(s)
- V V Emelyanov
- Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, 123098, Russia.
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35
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Cammisotto PG, Bukowiecki LJ, Deshaies Y, Bendayan M. Leptin biosynthetic pathway in white adipocytes. Biochem Cell Biol 2006; 84:207-14. [PMID: 16609702 DOI: 10.1139/o06-032] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum-Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.
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Affiliation(s)
- Philippe G Cammisotto
- Département de Pathologie et Biologie Cellulaire, Faculté de médecine, Université de Montréal, Canada
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Wazen RM, Tye CE, Goldberg HA, Hunter GK, Smith CE, Nanci A. In Vivo Functional Analysis of Polyglutamic Acid Domains in Recombinant Bone Sialoprotein. J Histochem Cytochem 2006; 55:35-42. [PMID: 16957163 DOI: 10.1369/jhc.6a7046.2006] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSPE) within an actual in vivo environment. Full-length prBSPE and prBSPE in which the poly [E] domains were replaced by polyalanine (prBSPA) were tagged with dinitrophenol (DNP). Tagged preparations comprised intact molecules and some fragmented forms. They were infused through a surgically created hole in the bone of rat hemimandibles and detected using immunogold labeling with anti-DNP antibodies. prBSPE-DNP was consistently immunodetected along exposed mineralized bone surfaces and osteocyte canaliculi at the surgical site. Few gold particles were observed on these surfaces when prBSPA-DNP was infused. Quantitative analyses showed significant differences in labeling between prBSPE-DNP (5.04 ± 0.73 particles/μm2) and prBSPA-DNP (1.37 ± 0.35 particles/μm2). These results indicate that poly [E] domains influence binding of prBSPE to surfaces presenting a mixture of mineral and proteins bathed by tissue fluids and suggest that they may similarly mediate the interaction of native BSP in the bone environment.
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Affiliation(s)
- Rima M Wazen
- Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Station Centre-Ville, Montreal, QC, Canada
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Mugabe C, Halwani M, Azghani AO, Lafrenie RM, Omri A. Mechanism of enhanced activity of liposome-entrapped aminoglycosides against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2006; 50:2016-22. [PMID: 16723560 PMCID: PMC1479138 DOI: 10.1128/aac.01547-05] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of > or =32 versus < or =8 microg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% +/- 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% +/- 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 +/- 2.3 versus 24.2 +/- 6.2 gold particles/bacterium, P < or = 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells.
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Affiliation(s)
- Clement Mugabe
- The Novel Drug and Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada
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Sané AT, Sinnett D, Delvin E, Bendayan M, Marcil V, Ménard D, Beaulieu JF, Levy E. Localization and role of NPC1L1 in cholesterol absorption in human intestine. J Lipid Res 2006; 47:2112-20. [PMID: 16829661 DOI: 10.1194/jlr.m600174-jlr200] [Citation(s) in RCA: 131] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum. Signal was also detected in lysosomes, endosomes, and mitochondria. Confirmation of cellular NPC1L1 distribution was obtained by immunocytochemistry. Knockdown of NPC1L1 caused a decline in the ability of Caco-2 cells to capture micellar [(14)C]free cholesterol. Furthermore, this NPC1L1 suppression resulted in increased and decreased mRNA levels and activity of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, and of ACAT, the key enzyme in cholesterol esterification, respectively. An increase was also noted in the transcriptional factor sterol-regulatory element binding protein that modulates cholesterol homeostasis. Efforts were devoted to define the impact of NPC1L1 knockdown on other mediators of cholesterol uptake. RT-PCR evidence is presented to show the significant decrease in the levels of scavenger receptor class B type I (SR-BI) with no changes in ABCA1, ABCG5, and cluster determinant 36 in NPC1L1-deficient Caco-2 cells. Together, our data suggest that NPC1L1 contributes to intestinal cholesterol homeostasis and possibly cooperates with SR-BI to mediate cholesterol absorption in humans.
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Affiliation(s)
- Alain Théophile Sané
- Department of Nutrition, CHU-Sainte-Justine, University of Montreal, Montreal, Quebec H3T 1C5, Canada
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Li YY, Zhu GY, Gao YX, Yang ZY, Wang ZY. Alteration of the pancreatic endocrine component in the early stage of acute necrotic pancreatitis in rats. ACTA ACUST UNITED AC 2006; 7:164-9. [PMID: 16808797 DOI: 10.1111/j.1443-9573.2006.00262.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
OBJECTIVE To investigate alterations of the pancreatic endocrine component in the early stage of acute necrotic pancreatitis (ANP) in rats. METHODS Thirty-six Sprague-Dawley rats were randomly allocated to two groups: ANP group (n = 18) and sham-operated (control) group (n = 18). ANP was induced by retrograde injection of 4% sodium deoxycholate (40 mg/kg, 0.1 mL/min) into the biliopancreatic duct and the severity of pancreatitis induced was assessed by histopathological examination and level of plasma amylase. The pancreatic endocrine function was assessed by measuring the levels of plasma glucose and insulin and by measuring the insulin content in pancreatic beta cells by immunofluorescence and immunocytochemistry. RESULTS Five hours after operation, the pancreas of rats in the ANP group showed pathological changes with edema, hemorrhage, fatty necrosis, acinar destruction and leukocyte infiltration in the exocrine portion of the pancreas. Plasma amylase activity increased significantly (P < 0.01) and bloody ascites appeared in the abdominal cavity. Nevertheless the endocrine islets appeared normal and the beta cells contained intensive labeling of insulin. Levels of glucose and insulin in plasma increased significantly. In the ANP group, 5 h after operation the plasma level of glucose was 8.18 +/- 2.26 mmol/L vs 6.39 +/- 1.26 mmol/L, and of insulin was 23.27 +/- 3.50 MIU/L vs 18.40 +/- 3.98 MIU/L. In the control group, 5 h after operation the plasma level of glucose was 9.39 +/- 0.62 mmol/L vs 5.89 +/- 0.62 mmol/L, and of insulin was 26.28 +/- 4.77 MIU/L vs 12.89 +/- 2.05 MIU/L; there was no significant difference between these two groups (P > 0.05). After a bolus injection of glucose, however, a much higher level of insulin was found in the control group (35.30 +/- 5.05 MIU/L) than that in the ANP group (23.91 +/- 4.62 MIU/L, P < 0.05). CONCLUSIONS There may be an impaired ability of insulin release in response to glucose stimulation in the early stage of ANP, although the morphology of the pancreatic endocrine component remains intact.
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Affiliation(s)
- Yong Yu Li
- Department of Pathophysiology, Medical School of Tongji University, Shanghai, China.
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Bendayan R, Ronaldson PT, Gingras D, Bendayan M. In situ localization of P-glycoprotein (ABCB1) in human and rat brain. J Histochem Cytochem 2006; 54:1159-67. [PMID: 16801529 PMCID: PMC3957801 DOI: 10.1369/jhc.5a6870.2006] [Citation(s) in RCA: 158] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
Abstract
Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.
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Affiliation(s)
- Reina Bendayan
- Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Canada.
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Boucher E, Mayer G, Londono I, Bendayan M. Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats. Kidney Int 2006; 69:1570-7. [PMID: 16541018 DOI: 10.1038/sj.ki.5000316] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.
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Affiliation(s)
- E Boucher
- Department of Pathology and Cell Biology, University of Montreal, Montreal, Quebec, Canada
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Allodi S, Bressan CM, Carvalho SL, Cavalcante LA. Regionally specific distribution of the binding of anti-glutamine synthetase and anti-S100 antibodies and of Datura stramonium lectin in glial domains of the optic lobe of the giant prawn. Glia 2006; 53:612-20. [PMID: 16435368 DOI: 10.1002/glia.20317] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
We previously characterized some crustacean glial cells by markers such as 2',3'-cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein. Here we use antibodies against glutamine synthetase full-length molecule (anti-GS/FL), a GS C-terminal peptide (anti-GS/20aa-C), and brain S100 (anti-S100), as well as the binding of the insect glia and rat astrocytic marker Datura stramonium lectin (DSL), in the optic lobe of the prawn Macrobrachium rosenbergii. All markers label the lamina ganglionaris cartridge region (lighter: anti-GS/FL; heavier: DSL). In addition, anti-GS/FL labels superficial somata of external and internal medullas and internal chiasm cells. Both anti-GS/20aa-C and anti-S100 label heavily the glial sheaths of the lamina ganglionaris. In addition, anti-S100 binds to the perineurial glia of medullary parenchymal vessels. Western blot analyses show that both anti-GS/FL and anti-GS/20aa-C bind mostly to a band of 50-55 kDa, compatible with a long isoform of vertebrate GS, and accessorily to a possible dimer and, in the case of anti-GS/20aa-C, to an ill-defined band of intermediate mass. Binding of anti-S100 is selective for a single band of about 68 kDa but shows no protein in the weight range of the canonical S100 protein superfamily. DSL reveals two bands of about 75 and about 120 kDa, thus within the range of maximal recognition for rat astrocytes. Our results suggest that phenotype protein markers of the optic lobe glia share antigenic determinants with S100 and (a long form of) GS and that, similarly to vertebrate and insect glia, crustacean glia protein and N-glycan residue markers display regional heterogeneity.
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Affiliation(s)
- Silvana Allodi
- Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
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Bosshardt DD, Sculean A, Donos N, Lang NP. Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins. Eur J Oral Sci 2006; 114 Suppl 1:225-31; discussion 254-6, 381-2. [PMID: 16674690 DOI: 10.1111/j.1600-0722.2006.00300.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern.
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Affiliation(s)
- Dieter D Bosshardt
- Department of Periodontology and Fixed Prosthodontics, School of Dental Medicine, University of Berne, Berne, Switzerland.
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Li YY, Bendayan M. Alteration of chaperonin60 and pancreatic enzyme in pancreatic acinar cell under pathological condition. World J Gastroenterol 2006; 11:7359-63. [PMID: 16437643 PMCID: PMC4723395 DOI: 10.3748/wjg.v11.i46.7359] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the changes of chaperonin60 (Cpn60) and pancreatic enzymes in pancreatic acinar cells, and to explore their roles in the development of experimental diabetes and acute pancreatitis (AP). METHODS Two different pathological models were replicated in Sprague-Dawley rats: streptozotocin-induced diabetes and sodium deoxycholate-induced AP. The contents of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were measured by quantitative immunocytochemistry. RESULTS The levels of Cpn60 significantly increased in diabetes, but decreased in AP, especially in the zymogen granules of the pancreatic acinar cells. The elevation of Cpn60 was accompanied with the increased levels of pancreatic lipase and chymotrypsinogen in diabetes. However, a decreased Cpn60 level was accompanied by high levels of lipase and chymotrypsinogen in AP. The amylase level was markedly reduced in both the pathological conditions. CONCLUSION The equilibrium between Cpn60 and pancreatic enzymes in the acinar cells breaks in AP, and Cpn60 content decreases, suggesting an insufficient chaperone capacity. This may promote the aggregation and autoactivation of the premature enzymes in the pancreatic acinar cells and play roles in the development of AP.
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Affiliation(s)
- Yong-Yu Li
- Department of Pathophysiology, Medical College of Tongji University, Shanghai 200092, China.
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Cloutier M, Gingras D, Bendayan M. Internalization and transcytosis of pancreatic enzymes by the intestinal mucosa. J Histochem Cytochem 2006; 54:781-94. [PMID: 16517974 DOI: 10.1369/jhc.5a6877.2006] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.
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Affiliation(s)
- Maryse Cloutier
- Department of Pathology and Cell Biology, University of Montreal, Montreal, Quebec, Canada
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46
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Cammisotto PG, Gingras D, Renaud C, Levy E, Bendayan M. Secretion of soluble leptin receptors by exocrine and endocrine cells of the gastric mucosa. Am J Physiol Gastrointest Liver Physiol 2006; 290:G242-9. [PMID: 16239400 DOI: 10.1152/ajpgi.00334.2005] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Leptin is a hormone secreted by the gastric mucosa into the lumen of the stomach. It is present in its intact form in the intestine where it regulates nutrient absorption and intestinal mucosa integrity. We have identified the binding protein that protects leptin from the harsh conditions of the gastric juice. Immunoprecipitations and Western blot analyses demonstrated that leptin is present in the gastric mucosa and the gastric juice, bound to a protein corresponding to the extracellular domain of the leptin receptor. In the absence of this soluble receptor, leptin is rapidly degraded. Immunocytochemistry on rat gastric mucosa identified the cells and intracellular compartments involved in secretion of this complex. Leptin receptor extracellular domain and leptin are present along the rough endoplasmic reticulum-Golgi-granules secretory pathways and form a complex in the secretory granules of Chief and specific endocrine cells. The long-form membrane leptin receptor OB-Rb, the protease activator furin, and proprotein convertase 7 were found in Chief cell granules but not in those of endocrine cells. The shedding of the receptor occurs in the immature granules. It is concluded that in the immature secretory granules of Chief cells, furin activates proprotein convertase 7 that, in turn, cleaves the extracellular portion of membrane-bound leptin receptors. Leptin bound to its soluble receptor forms a complex that is resistant to the gastric juice. Endocrine cells, on the other hand, generate a soluble leptin receptor by mechanisms different from those of the exocrine cells.
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Affiliation(s)
- Philippe G Cammisotto
- Département de Pathologie et Biologie Cellulaire, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal, Québec, Canada H3C 3J7
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Massa LF, Bradaschia-Correa V, Arana-Chavez VE. Immunocytochemical Study of Amelogenin Deposition during the Early Odontogenesis of Molars in Alendronate-treated Newborn Rats. J Histochem Cytochem 2006; 54:713-25. [PMID: 16461365 DOI: 10.1369/jhc.5a6853.2006] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.
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Affiliation(s)
- Luciana F Massa
- Laboratory of Mineralized Tissue Biology, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, SP, Brazil
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Joly E, Bendayan M, Roduit R, Saha AK, Ruderman NB, Prentki M. Malonyl-CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes. FEBS Lett 2005; 579:6581-6. [PMID: 16298369 DOI: 10.1016/j.febslet.2005.10.050] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2005] [Accepted: 10/25/2005] [Indexed: 10/25/2022]
Abstract
A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.
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Affiliation(s)
- Erik Joly
- Molecular Nutrition Unit and the Montreal Diabetes Research Center, Centre de recherche du CHUM, Pavillon de Sève, Y-4603, 1560 Sherbrooke Est, and the Department of Nutrition and Biochemistry, Université de Montréal, Montréal PQ, Canada, H3T 1C5
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Sponner A, Unger E, Grosse F, Weisshart K. Differential polymerization of the two main protein components of dragline silk during fibre spinning. NATURE MATERIALS 2005; 4:772-5. [PMID: 16184170 DOI: 10.1038/nmat1493] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2005] [Accepted: 08/10/2005] [Indexed: 05/04/2023]
Abstract
Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3, 6, 7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk.
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Affiliation(s)
- Alexander Sponner
- Institute for Molecular Biotechnology, Jena, Federal Republic of Germany
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Cammisotto PG, Renaud C, Gingras D, Delvin E, Levy E, Bendayan M. Endocrine and exocrine secretion of leptin by the gastric mucosa. J Histochem Cytochem 2005; 53:851-60. [PMID: 15995144 DOI: 10.1369/jhc.5a6620.2005] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.
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Affiliation(s)
- Philippe G Cammisotto
- Département de Pathologie et Biologie Cellulaire, Université de Montréal, C.P. 6128, Succursale Centre Ville, Montréal, Québec, Canada H3C 3J7
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