bluuebunny/crossref_metadata_2025 huggingface dataset:
A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
Authors: Roberg Karin , Öllinger Karin
Published: April 2011

We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.
Ultrastructural Localization of Glycolytic Enzymes on Sarcoplasmic Reticulum Vesicles
Authors: Xu Kai Y. , Becker Lewis C.
Published: April 2011

We have previously obtained indirect evidence that sarcoplasmic reticulum (SR) vesicles from cardiac and skeletal muscle contain the complete chain of glycolytic enzymes from aldolase to pyruvate kinase. To investigate directly whether pyruvate kinase and other glycolytic enzymes are anatomically associated with the SR, electron microscopic immunogold labeling studies were carried out in isolated SR vesicles using specific primary antibodies against selected glycolytic enzymes and Ca2+-ATPase, and appropriate secondary antibodies labeled with 6-nm or 12-nm gold particles. Pyruvate kinase was broadly dispersed on the cytoplasmic side of the SR membrane of both cardiac and skeletal muscle vesicles. With 6-nm gold particles, the density of binding to pyruvate kinase was 2522 ± 445 and 4171 ± 1379 particles/μm2 for cardiac and skeletal muscle SR, respectively. Binding densities to Ca2+-ATPase were similar (2550 ± 639 particles/μm2 for cardiac SR, 3877 ± 408 particles/μm2 for skeletal muscle SR). Immunogold labeling of ultrathin sections indicated that pyruvate kinase was attached to the SR membrane and located immediately adjacent to the Ca2+-ATPase. Aldolase and glyceraldehyde phosphate dehydrogenase were also found to be attached to the cytoplasmic side of SR vesicles and located in close proximity to Ca2+-ATPase. These results provide the first ultrastructural evidence that glycolytic enzymes are anatomically associated with SR membranes and located near the SR Ca2+-ATPase. The results further support the hypothesis that ATP generated by SR-associated glycolytic enzymes is coupled to SR Ca2+ active transport.
Human TIMP-3 Is Expressed During Fetal Development, Hair Growth Cycle, and Cancer Progression
Authors: Airola Kristiina , Ahonen Matti , Johansson Nina , Heikkilä Päivi , Kere Juha , Kähäri Veli-Matti , Saarialho–Kere Ulpu K.
Published: April 2011

We studied the expression and regulation of TIMP-3, a recently cloned member of the tissue inhibitor of the metalloproteinase family, during human fetal development and in various human tissues, with emphasis on epithelial structures. Expression of TIMP-3 mRNA was detected by in situ hybridization in developing bone, kidney, and various mesenchymal structures. At 16 weeks of gestation, ectoderm-derived cells of hair germs expressed TIMP-3 mRNA, and beginning from the twentieth week consistent expression was detected in epithelial outer root sheath cells of growing hair follicles. In normal adult human skin, expression of TIMP-3 mRNA was limited to hair follicles, starting at the early anagen (growing) phase and vanishing at the catagen (regressing) phase. TIMP-3 mRNA was not detected in benign hair follicle-derived tumors but was present in tumor cells of infiltrative basal cell carcinomas and in surrounding stromal cells in squamous cell carcinomas. Human primary keratinocytes in culture expressed TIMP-3 mRNAs, the levels of which were upregulated by transforming growth factor-β (TGF-β), whereas interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) had no effect. Our results suggest a role for TIMP-3 in connective tissue remodeling during fetal development, hair growth cycle, and cancer progression.
Epitopes of Components of the Plasminogen Activation System are Re-exposed in Formalin-fixed Paraffin Sections by Different Retrieval Techniques
Authors: Ferrier Cilia M. , van Geloof Winny L. , de Witte Hans H. , Kramer Michael D. , Ruiter Dirk J. , van Muijen Goos N.P.
Published: April 2011

We present a systematic analysis of the sensitivity and specificity of immunohistochemical stainings for components of the plasminogen activation system, i.e., uPA, tPA, PAI-1, PAI-2, and uPAR, on routinely processed (formalin-fixed, paraffin-embedded) tissues. Five to nine antibodies per component were tested and the influence of different antigen retrieval regimens on immunoreactivity was investigated. We studied six different microwave-mediated pretreatments and two pretreatments by proteolytic digestion. First, positive and negative control tissues were stained. Then, frozen and paraffin sections from the same cancer lesions were stained after specific modes of pretreatment and with selected antibodies. For each component, one or a few of the tested Abs gave optimal staining on paraffin sections when combined with a particular tissue pretreatment. For PAI-1, and to a lesser degree also for tPA, an underrepresentation of stromal cell staining in paraffin material was found, whereas tumor cells showed good staining. For uPA, PAI-2, and uPAR, consistent staining results were obtained on paraffin sections.
Immunohistochemistry of Carbonic Anhydrase Isozyme IX (MN/CA IX) in Human Gut Reveals Polarized Expression in the Epithelial Cells with the Highest Proliferative Capacity
Authors: Saarnio Juha , Parkkila Seppo , Parkkila Anna-Kaisa , Waheed Abdul , Casey Matthew C. , Zhou Xiao Yan , Pastoreková Silvia , Pastorek Jaromir , Karttunen Tuomo , Haukipuro Kari , Kairaluoma Matti I. , Sly William S.
Published: April 2011

MN/CA IX is a recently discovered member of the carbonic anhydrase (CA) gene family that has been identified in the plasma membranes of certain tumor and epithelial cells and found to promote cell proliferation when transfected into NIH3T3 cells. This study presents localization of MN/CA IX in human gut and compares its distribution to those of CA I, II, and IV, which are known to be expressed in the intestinal epithelium. The specificity of the monoclonal antibody for MN/CA IX was confirmed by Western blots and immunostaining of COS-7 cells transfected with MN/CA IX cDNA. Immunohistochemical stainings of human gut revealed prominent polarized staining for MN/CA IX in the basolateral surfaces of the enterocytes of duodenum and jejunum, the reaction being most intense in the crypts. A moderate reaction was also seen in the crypts of ileal mucosa, whereas the staining became generally weaker in the large intestine. The results indicate isozyme-specific regulation of MN/CA IX expression along the cranial–caudal axis of the human gut and place the protein at the sites of rapid cell proliferation. The unique localization of MN/CA IX on the basolateral surfaces of proliferating crypt enterocytes suggests that it might serve as a ligand or a receptor for another protein that regulates intercellular communication or cell proliferation. Furthermore, MN/CA IX has a completely conserved active site domain of CAs suggesting that it could also participate in carbon dioxide/bicarbonate homeostasis.
Different Pattern of MRP Localization in Ciliated and Basal Cells from Human Bronchial Epithelium
Authors: Bréchot Jeanne-Marie , Hurbain Ilse , Fajac Anne , Daty Nicole , Bernaudin Jean-François
Published: April 2011

The MRP (multidrug resistance-associated protein) transmembrane transporter, which actively transports a wide variety of lipophilic substrates out of cancer cells, has been suggested to play a major role in cell detoxification via efflux of glutathione conjugates. Because bronchial epithelial cells are constantly exposed to environmental pollutants, MRP might be a particularly important defense mechanism against xenobiotics. This study was therefore designed to investigate MRP localization by immunohistochemistry in bronchial epithelial cells collected by scraping from surgical specimens. In parallel, MRP mRNA was detected by reverse transcriptase chain reaction (rt-PCR) in bronchial cell lysates. However, the pattern of protein expression differed markedly according to cell type. In ciliated epithelial cells, immunostaining was restricted to the basolateral surface, without any labeling at the apical surface, which is at variance with the localization of CFTR and MDR1 proteins, other members of the same family of transporters. In basal cells, MRP was present over the entire circumference of the plasma membrane. Basal cells were identified by their morphology and specifically after incubation with an anticytokeratin 17 monoclonal antibody. In conclusion, the different patterns of localization suggest specific roles for MRP in basal and ciliated cells.
Tyramide Amplification Allows Anterograde Tracing by Horseradish Peroxidase-conjugated Lectins in Conjunction with Simultaneous Immunohistochemistry
Authors: Kressel Michael
Published: April 2011

Current protocols for a combined approach of anterograde tracing with carbocyanine dyes or horseradish peroxidase (HRP) conjugates and immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was investigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA–HRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGA–HRP into the nodose ganglion of rats. By use of tyramide–biotin amplification, a dense fiber plexus of vagal afferents was visualized centrally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, large- and small-caliber vagal fibers and terminals could be demonstrated comparably to conventional tracing techniques using carbocyanine dyes or WGA–HRP and TMB histochemistry. Combination with immunohistochemistry could easily be done, requiring only one more incubation step, and did not result in loss of sensitivity of the tracing. With this method and con-focal microscopy, the presence of Ca binding proteins in vagal afferent terminals could be demonstrated. Tyramide amplification allows sensitive anterograde tracing with low background staining in conjunction with immunohistochemistry of intra-axonal markers.
A New Method for Intense Staining of Myelin
Authors: McNally Karen J. , Peters Alan
Published: April 2011

We describe a new method for intense staining of myelin. The stain involves immersing frozen or vibratome sections in 4% normal horse serum. A DAB reaction is then carried out, which results in the deposition of reaction product in myelin sheaths. On intensification of this reaction product using the silver enhancement technique described by Görcs, myelin stains an intense black color, making the preparations suitable for photography. The stain is especially useful for determining the distribution of myelinated fibers in gray matter.
Earliest Steps in Primary Tumor Formation and Micrometastasis Resolved with Histochemical Markers of Gene-tagged Tumor Cells
Authors: Culp L. A. , Lin W.-c. , Kleinman N. R. , O'Connor K. L. , Lechner R.
Published: April 2011

To facilitate detection of tumor cells at the highest resolution in any organ in athymic nude mouse model systems, a histochemical marker gene [bacterial IacZ or human placental alkaline phosphatase (ALP)] was transfected into specified transformed/tumor cells (fibrosarcoma or neuroblastoma). The fates of tumor cells were followed qualitatively and quantitatively by histochemical staining of whole organs or organ sections. Primary tumors developed initially via formation of “curly-haired” complexes of cells in the subcutis or dermis, followed by division of a large fraction of cells. When two tumor classes were mixed before injection, outgrowth occurred in regional concentrations of the primary tumor. Blood microvessels were detectable within 72 hr of injection, growing into tumor regions. IV injection routinely yielded multicellular foci in the lungs within minutes as precursors of experimental metastases. Micrometastasis was further resolved with cells “inactivated” by different treatments and by co-injection of two different tagged cell types. These approaches using different histochemical marker genes to “tag” different tumor cell classes, along with more advanced molecular biological approaches, permit us to characterize gene expression and its reversibility during the earliest stages of primary tumor formation and micrometastasis to virtually any organ in the recipient animal.
An Essential Role for the Interaction Between Hyaluronan and Hyaluronan Binding Proteins During Joint Development
Authors: Dowthwaite Gary P. , Edwards Jo C. W. , Pitsillides Andrew A.
Published: April 2011

We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HA-HABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HA-HABP interactions play a major role in the initial separation events occurring during joint formation.
Detection and Localization by In Situ Molecular Biology Techniques and Immunohistochemistry of Hepatitis C Virus in Livers of Chronically Infected Patients
Authors: Walker Francine M. , Dazza Marie-Christine , Dauge Marie-Christine , Boucher Olivier , Bedel Christophe , Henin Dominique , Lehy Therese
Published: April 2011

Hepatitis C virus (HCV) detection in the livers of chronically infected patients remains a debatable issue. We used immunohistochemistry, in situ hybridization (ISH) alone or after microwave heating with FITC-labeled probes, RT-PCR with unlabeled primers followed by ISH (RT-PCR-ISH), and in situ RT-PCR with FITC-labeled primers (in situ RT-PCRd) to localize the virus in 38 liver biopsy specimens from 21 chronically infected HCV patients treated with interferon-α (IFN-α). Biopsies were taken at the beginning and end of IFN-α treatment and 1 year later. Results were compared with that of HCV-PCR in serum. RT-PCR-ISH and in situ RT-PCRd showed HCV signal in all liver biopsies even in responders with seronegative HCV PCR. This signal was intranuclear, diffuse, or peripheral, in hepatocytes, bile ductule cells, and lymphocytes. Cytoplasmic signals were occasionally observed. Whereas the percentage of labeled hepatocytes remained constant, the number of labeled lymphoid follicles decreased after INF-α therapy. Immunohistochemistry resulted in the same pattern of positivity but it was weaker and inconstant. This study indicates the persistency of HCV latency in IFN-α responders 1 year after IFN-α treatment cessation, a finding that certainly deserves confirmation.
Acinar Cells Are Target Cells for Androgens in Mouse Submandibular Glands
Authors: Señorale-Pose Mario , Jacqueson Arnaud , Rougeon François , Rosinski-Chupin Isabelle
Published: April 2011

The variable coding sequence (VCS) multigene family encodes diverse salivary proteins, such as the SMR1 prohormone and the PR-VB1 proline-rich protein in the rat. In situ hybridization was used to study the cell-specific expression of two new mouse VCS genes, Vcs1 and Vcs2. We show that the Vcs1 transcripts, which code for a proline-rich protein, MSG1, are highly abundant in male and female parotid glands, in which they are specifically detected in acinar cells. No expression was seen in the submandibular or sublingual glands. In contrast, Vcs2 transcripts were found only in the acinar cells of the submandibular glands (SMGs) of male mice, in which they are expressed in response to androgens. Expression was found to be heterogeneous within acinar structures. No Vcs2 transcripts were detected in the SMGs of females or castrated males by Northern blot, RNase protection, or in situ hybridization. Androgen administration to females or castrated males induced expression at a level comparable to that of intact males. The Vcs2 gene is the first example of a mouse androgen-regulated gene that is expressed in SMG acinar cells. This result, in addition to our previous observation on SMR1 expression in rats, demonstrates that both acinar cells and granular convoluted tubule (GCT) cells are target cells for androgen action in rodent SMG.
Immunocytochemical and In Situ Hybridization Studies of the Distribution of Calbindin D9k in the Bovine Placenta Throughout Pregnancy
Authors: Nikitenko Leonid , Morgan Geoffrey , Kolesnikov Sergei I. , Wooding F.B. Peter
Published: April 2011

The fetus must transport considerable and increasing amounts of calcium across the placental trophoblast epithelium to support growth and development and bone formation. Active calcium transport across epithelia has been shown to correlate with calbindin D9k or 28k content. This study examined the distribution of calbindin D9k (9CBP) protein and mRNA during pregnancy in the bovine placenta to determine its possible role in calcium transport in this system. The immunocytochemical results show 9CBP in an increasing percentage of interplacentomal uninucleate trophoblast cells until, at term, all show a level at least eight times that of any other placental cell. There is a similar, although smaller, rise in their 9CBP mRNA content. The mature interplacentomal binucleate cell (∼5% of the total) contains no 9CBP at any stage of pregnancy. In interplacentomal uterine epithelium, 9CBP protein and mRNA decrease to zero in late pregnancy but the glands maintain constant low levels throughout. In the placentome trophoblast, uninucleate cells show insignificant amounts but binucleate cells (15–20% of the total trophoblast cells) contain considerable levels of both 9CBP protein and mRNA, as do all the uninucleate uterine epithelial cells. The placentomal binucleate cells show peak values at mid-pregnancy; the placentomal uterine epithelium shows only small changes in levels in the second half of pregnancy. Increase in fetal calcium demand in the second half of pregnancy therefore correlates with a major increase in 9CBP only in the interplacentomal trophoblast, as we have also shown in the sheep and goat, indicating an important role for this region in active calcium transport by the ruminant placenta. The 9CBP is distributed uniformly in the cytosol and nucleoplasm, supporting a role in facilitated diffusion of calcium through the cell rather than a vesicular shuttle system.
Ribonuclease-Gold Labels Heparin in Human Mast Cell Granules: New Use for an Ultrastructural Enzyme Affinity Technique
Authors: Dvorak Ann M. , Morgan Ellen S.
Published: April 2011

We evaluated an enzyme affinity-gold ultrastructural technique designed to identify RNA-rich structures, based on an RNase-gold (R-G) probe in human mast cells (HMCs). As expected, the R-G technique labeled RNA-containing ribosomes and nucleoli in HMCs. The heparin-rich secretory granules in HMCs were also labeled. Extensive studies revealed that HMCs isolated from lung or skin and sustained in short-term cultures, derived de novo in growth factor-supplemented cord blood cell cultures, or present in vivo in multiple sites all shared this property. We performed a large number of controls designed to examine the HMC granule binding characteristics of gold alone, of irrelevant protein- or enzyme-gold reagents, of the role of charge and enzyme activity after various enzyme digestions, after blocking with macromolecules, after exposure to inhibitors of RNase, of heparin, or to irrelevant enzyme inhibitors, including staining of macromolecule-containing test agar blocks and a variety of combined absorption and digestion experiments of the binding of R-G to HMC granules. These studies established that the R-G method detected heparin in this site in conventionally prepared, well-preserved electron microscopic samples. These findings demonstrate a new use for this enzyme affinity-gold technique in mast cell biology, based on the known property of heparin as an inhibitor of RNase.
Rat Endocrine Pancreatic Development in Relation to Two Homeobox Gene Products (Pdx-1 and Nkx 6.1)
Authors: Øster Anne , Jensen Jan , Serup Palle , Galante Philip , Madsen Ole D. , Larsson Lars-Inge
Published: April 2011

We studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more β-cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1- and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.
Homeobox Gene Product Nkx 6.1 Immunoreactivity in Nuclei of Endocrine Cells of Rat and Mouse Stomach
Authors: Øster Anne , Jensen Jan , Edlund Helena , Larsson Lars-Inge
Published: April 2011

The homeobox gene product Nkx 6.1 is of unknown function but is expressed in the pancreas and the antropyloric mucosa of the stomach. In the adult pancreas, Nkx 6.1 possesses an insulin cell-restricted distribution, whereas its localization in the stomach is unknown. We now show that the vast majority of serotonin-producing enterochromaffin cells of the antropyloric mucosa contain Nkx 6.1-immunoreactive nuclei. In addition, a subpopulation of cells co-storing serotonin and gastrin display Nkx 6.1-positive nuclei. Such cells have been postulated to represent precursors of mature gastrin and serotonin cells. The nuclei of the co-storing cells have previously also been found to be positive for another homeodomain protein, Pdx-1. Pdx-1-deficient animals were therefore investigated and were found to be devoid of Nkx 6.1-positive nuclei. Our data show that Pdx-1 is needed for Nkx 6.1 expression and suggest a role for Nkx 6.1 in the maturation of gastrin- and serotonin-positive precursor cells.
Cytochemical Demonstration of Oxidative Damage in Alzheimer Disease by Immunochemical Enhancement of the Carbonyl Reaction with 2,4-Dinitrophenylhydrazine
Authors: Smith Mark A. , Sayre Lawrence M. , Anderson Vernon E. , Harris Peggy L.R. , Beal M. Flint , Kowall Neil , Perry George
Published: April 2011

Formation of carbonyls derived from lipids, proteins, carbohydrates, and nucleic acids is common during oxidative stress. For example, metal-catalyzed, “site-specific” oxidation of several amino acid side-chains produces aldehydes or ketones, and peroxidation of lipids generates reactive aldehydes such as malondialdehyde and hydroxynonenal. Here, using in situ 2,4-dinitrophenylhydrazine labeling linked to an antibody system, we describe a highly sensitive and specific cytochemical technique to specifically localize biomacromolecule-bound carbonyl reactivity. When this technique was applied to tissues from cases of Alzheimer disease, in which oxidative events including lipoperoxidative, glycoxidative, and other oxidative protein modifications have been reported, we detected free carbonyls not only in the disease-related intraneuronal lesions but also in other neurons. In marked contrast, free carbonyls were not found in neurons or glia in age-matched control cases. Importantly, this assay was highly specific for detecting disease-related oxidative damage because the site of oxidative damage can be assessed in the midst of concurrent age-related increases in free carbonyls in vascular basement membrane that would contaminate biochemical samples subjected to bulk analysis. These findings demonstrate that oxidative imbalance and stress are key elements in the pathogenesis of Alzheimer disease.
A Histochemical Approach to Correlative Light and Electron Microscopic Detection of Acidic Glycoconjugates by a Sensitized High Iron Diamine Method
Authors: Hirabayashi Yoshifumi , Yamada Kazuyori
Published: April 2011

A sensitized high iron diamine method is among the reliable and useful histochemical means of detecting acidic glycoconjugates by light microscopy. Because the final reaction products obtained using this method are heavy metals, it can be applied to specimens for visualization by both light and electron microscopy. In this study the high iron diamine method was utilized successfully as a correlative light and electron microscopic method for detection of acidic glycoconjugates.
Staining of the Midbody by an Anti-digoxin-specific Antibody
Authors: Dirks Roeland W. , Raap Anton K.
Published: April 2011

Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody. This was confirmed by double staining of cells with anti-digoxin and antia -tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody.
Distribution of Integrins During Human Fetal Lung Development
Authors: Coraux Christelle , Delplanque Aurélie , Hinnrasky Jocelyne , Peault Bruno , Puchelle Edith , Gaillard Dominique
Published: April 2011

Interactions between epithelial cells and the extracellular matrix through integrins play a key role in the development of the lung by modulating branching morphogenesis, epithelial cell polarization, and differentiation. To determine the role of integrins during the different stages of lung development, we investigated the distribution of eight integrin subunits in the trachea and lung from human fetuses. In distal airways, during the early pseudoglandular stage of development, the α2-, α5-, α6-, αv-, and β1-subunits were detected in all epithelial cell plasma membranes, and polarized but undifferentiated tracheal epithelial cells expressed α3-, α6-, and β1-subunits in the plasma membrane of the cells facing the basement membrane. The α6- and β4-chains were detected along the basal plasma membrane of the basal cells in differentiated tracheal epithelia. The α4-subunit was detected in all respiratory cells throughout fetal development. In the submucosal glands, myoepithelial cells expressed the integrin subunits found in the undifferentiated cells of the developing airways, whereas the secretory cells expressed only α2-, α3-, α4-, α6-, and β1-subunits. These results demonstrate differential expression of integrins during lung development and suggest that integrins may play multiple roles in organogenesis and maturation of respiratory surface epithelium and glands.
Membrane Dipeptidase in the Pig Exocrine Pancreas: Ultrastructural Localization and Secretion
Authors: LeBel Denis , Grondin Gilles , Cook Sarah , Hooper Nigel M.
Published: April 2011

The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.
Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method
Authors: Moreira Jorge E. , Dodane Valerie , Reese Thomas S.
Published: April 2011

The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.
Localization of Carbonic Anhydrase IV in Rat and Human Heart Muscle
Authors: Sender Susanne , Decker Brigitte , Fenske Christiane D. , Sly William S. , Carter Nicholas D. , Gros Gerolf
Published: April 2011

We investigated carbonic anhydrase IV (CA IV) in rat and human heart with immunohistochemical methods by both light and electron microscopy. In cryosections that were incubated with anti-CA IV/FITC, the capillaries showed a strong reaction for CA IV. In paraffin and semithin sections treated with anti-CA IV/ABC (avidin-biotin-peroxidase complex) blood vessels, capillaries, and sarcolemma (SL) were positively stained. By staining ultrathin sections with anti-CA IV/immunogold, CA IV could also be demonstrated at the latter two locations, including the specialized sarcolemmal structures intercalated discs, and T-tubules. In addition, by this method CA IV was seen to be associated with the sarcoplasmic reticulum (SR). The absence of immunostaining in SR and/or SL with some techniques probably indicates a problem of accessibility of the antigenic sites. In line with the immunohistochemical results, CA IV mRNA expression was visualized in both endothelial and muscle cells by in situ hybridization histochemistry.
Intranuclear Distribution of HMGI/Y Proteins: An Immunocytochemical Study
Authors: Martelli Alberto M. , Riccio Massimo , Bareggi Renato , Manfioletti Guidalberto , Tabellini Giovanna , Baldini Giovanna , Narducci Paola , Giancotti Vincenzo
Published: April 2011

The intranuclear distribution of HMGI/Y proteins was analyzed by immunofluorescent staining in several cell lines using a polyclonal antibody that stained a fibrogranular network. In actively growing 3T3 fibroblasts, HMGI/Y proteins were mainly localized to heterochromatin masses, whereas in quiescent cells they were more diffusely distributed. Double labeling experiments showed a co-localization of HMGI/Y with DNA topoisomerase IIα. These results are in agreement with previously published biochemical data and indicate a possible involvement of HMGI/Y proteins in several nuclear functions, including chromatin organization and gene expression.
Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy
Authors: Gatti Rita , Belletti Silvana , Orlandini Guido , Bussolati Ovidio , Dall'Asta Valeria , Gazzola Gian Carlo
Published: April 2011

SUMMARY Although morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.
Temporal-Spatial Distribution of Hepatocyte Nuclear Factor-3β in Developing Human Lung and Other Foregut Derivatives
Authors: Stahlman Mildred T. , Gray Mary E. , Whitsett Jeffrey A.
Published: April 2011

SUMMARY We assessed the temporal-spatial distribution of hepatocyte nuclear factor-3β (HNF-3β) in developing human lung and other foregut derivatives. Tissue from 31 fetuses (10-40 weeks) and 24 infants with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD) (2 days to 7 months) was studied. HNF-3β was detected in nuclei of epithelial cells of trachea and of conducting and terminal airways at 10 weeks. Thereafter, epithelial nuclei were immunolabeled more widely in peripheral than proximal airways. HNF-3β was confined to bronchiolo-alveolar portals and Type II cells in nonfetal lung. In infants with BPD, HNF-3 β was expressed abundantly in regenerating epithelial cells at the periphery of lung lobules. HNF-3 β was also detected in fetal esophagus, pancreas, duodenum, stomach, and gallbladder, suggesting that it is a marker for progenitor cells in foregut derivatives. The pattern of expression of HNF-3 β in the lung was similar to that of thyroid transcription factor-1 (TTF-1) at all ages. The temporal-spatial patterns of HNF-3 β and TTF-1 in the developing and regenerating lung are consistent with their proposed role in epithelial cell differentiation, regeneration, and surfactant protein gene expression.
Cytochemical Localization of Ouabain-sensitive, K+-dependent p-Nitrophenylphosphatase Activity in the Choroid Plexus of Normal and Reserpinized Guinea Pigs
Authors: Dai Chun-Fu , Kanoh Naoyuki
Published: April 2011

SUMMARY High doses of reserpine induce depletion of biogenic amines. The K-NPPase activity of choroid plexus was determined after one-shot reserpine administration using cerium-based cytochemistry. In normal untreated animals, reaction product was found on the microvilli of the choroidal epithelium but was almost undetectable 3 and 7 days after reserpinization. At 20 days after reserpinization, however, it was detectable. These findings suggested that reserpine decreased the choroidal Na,K-ATPase activity, and that catecholamines might be essential to maintain normal choroidal Na,K-ATPase activity.
The Size of Sites Containing SR Proteins in Human Nuclei: Problems Associated with Characterizing Small Structures by Immunogold Labeling
Authors: Iborra Francisco J. , Cook Peter R.
Published: April 2011

Some SR proteins are associated with eukaryotic transcripts as they move from synthetic sites (transcription “factories”), through downstream sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of ∼200 S (diameter ∼50 nm). In ultrathin sections of HeLa nuclei, indirect immunogold labeling with a specific antibody gives many small clusters of ∼10 gold particles (diameter 50–80 nm). We gauged errors in estimating the diameter of underlying structures marked by immunogold probes (lengths ∼20 nm). We examined systematically how probe dimensions affected cluster diameter. Probes contained one to three immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 5–15 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster diameter by 10 nm, (c) reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by ∼4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by ∼7 nm and led to a peripheral concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying structures had diameters of 50 nm, we find that errors ranged from −20% to + 50%.
Human Corneal Epithelial Basement Membrane and Integrin Alterations in Diabetes and Diabetic Retinopathy1
Authors: Ljubimov Alexander V. , Huang Zhi-shen , Huang Gang H. , Burgeson Robert E. , Miner Jeffrey H. , Gullberg Donald , Ninomiya Yoshifumi , Sado Yoshikazu , Kenney M. Cristina
Published: April 2011

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (α1β1γ1) and laminin-10 (α5β1γ1) was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for α3β1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.
Expression of Gross Cystic Disease Fluid Protein-15/Prolactininducible Protein in Rat Salivary Glands
Authors: Mirels Lily , Hand Arthur R. , Branin Holly J.
Published: April 2011

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20–22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.
Simultaneous Visualization of the Yellow and Green Forms of the Green Fluorescent Protein in Living Cells
Authors: Baumann Chris T. , Lim Carol S. , Hager Gordon L.
Published: April 2011

In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a redshifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476–nm and 488–nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.
Electron Microscopic Visualization of Receptor-mediated Endocytosis of DiI-labeled Lipoproteins by Diaminobenzidine Photoconversion
Authors: Dantuma Nico P. , Pijnenburg Marian A. P. , Diederen Jacques H. B. , Horst Dick J. Van der
Published: April 2011

We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells that had been incubated with DiI-labeled lipoproteins yielded a light- and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of DiI-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of DiI was not affected by aminotriazole. We conclude that specific staining of DiI-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here.
Nuclear Detection of Cellular Retinoic Acid Binding Proteins I and II with New Antibodies
Authors: Gaub Marie-Pierre , Lutz Yves , Ghyselinck Norbert B. , Scheuer Isabelle , Pfister Véronique , Chambon Pierre , Rochette-Egly Cécile
Published: April 2011

Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.
Ultrastructural Immunolocalization of Basic Fibroblast Growth Factor in Mast Cell Secretory Granules: Morphological Evidence for bFGF Release Through Degranulation
Authors: Qu Zhenhong , Kayton Robert J. , Ahmadi Proochista , Liebler Janice M. , Powers Michael R. , Planck Stephen R. , Rosenbaum James T.
Published: April 2011

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.