bluuebunny/crossref_metadata_2025 huggingface dataset:
Semi-automated Method of Quantifying Vasculature of 1-Methyl-1-nitrosourea-induced Rat Mammary Carcinomas Using Immunohistochemical Detection
Authors: McGinley John N. , Knott Katrina K. , Thompson Henry J.
Published: April 2011

Studies of the vascularization of autochthonous rodent mammary tumors are limited in number, and the majority have used Factor VIII staining for blood vessel detection. Moreover, little effort has been directed at measuring the vascularization of tissue immediately adjacent to a tumor despite its central importance in the process of angiogenesis. Thirty-six chemically-induced mammary carcinomas and tissue immediately adjacent to these carcinomas were used to develop a census counting method for quantitative assessment of intra- and extra-tumor vascularization. Blood vessels were identified using antiserum directed against either CD31 or Factor VIII. Techniques used to create digitized images of all tumors and the semi-automated methods for circumscribing the extra-tumoral region are described. For Factor VIII, CD31 allowed greater discrimination of blood vessels with areas <25 μm2 and demonstrated crisp staining of blood vessels, with minimal background and excellent preservation of tissue architecture. Census counting data support the use of CD31 for quantifying both intra- and extra-tumoral vascularization. This method provides a basis for standardizing the approach to evaluation of experimentally induced premalignant and malignant mammary lesions in rodent model systems used to investigate potential anti-angiogenic cancer preventive agents.
Immunochemical Demonstration of αs1- and β-Casein in Mouse Mammary Glands at Early Stages of Pregnancy
Authors: Kanazawa Takuya , Kohmoto Kaoru
Published: April 2011

We generated monoclonal antibodies (MAbs) against mouse αs1- and β-casein and used them to survey casein immunochemically in mammary glands of mice at pericoitous and pregnant stages. Two MAb-producing hybridoma cells, designated MCα1 cell and MCβ1 cell, were established. Each antibody, when used in Western blotting, recognized specifically mouse αs1- and β-casein among a wide spectrum of proteins of both a lactating mammary homogenate and mouse skim milk. Immunohistochemistry revealed αs1- and β-casein in sections of lactating mammary glands. Staining was found in substances in the lumen and cytoplasm of duct and alveolar cells, particularly in rough endoplasmic reticulum and the Golgi apparatus. Mammary glands at Days 2, 4, 6, 8, and 14 of pregnancy showed positive staining specific to both αs1- and β-casein in the lumen and cytoplasm of duct cells, whereas the glands at estrus and Day 0 of pregnancy were positive mainly for αs1-casein. Semiquantitative Western blotting analysis of both casein components in epithelial cell fractions from glands during pregnancy confirmed that intra-epithelial αs1- and β-casein changed during three phases, elevated from trace levels to detectable levels during initial stages of pregnancy (Days 0, 2, and 4), declined to lower levels during mid-pregnancy (Days 6 and 8), and then rose to high levels during late pregnancy (Day 14).
Immunohistochemical Localization of Angiotensin II Receptor and Local Renin-Angiotensin System in Human Colonic Mucosa
Authors: Hirasawa Koki , Sato Yoshihiko , Hosoda Yoshisuke , Yamamoto Tastuo , Hanai Hiroyuki
Published: April 2011

Angiotensin II (Ang II) regulates water and sodium transport in renal tubules and gastrointestinal tract. Two types of Ang II receptors have been cloned, but their distributions have not been determined in human colon. In addition, tissue renin-angiotensin systems (RAS) are believed to exist and to regulate local actions in human colon. We studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) the presence and localization of Ang II receptors Type 1 (AT1), Type 2 (AT2), and RAS components [angiotensinogen, renin, and angiotensin-converting enzyme (ACE)] in normal human colon. AT1 receptors were localized in vessel walls, myofibroblasts, macrophages, and surface epithelium. AT2 receptors were found in mesenchymal cells and weakly in parts of surface epithelium. Renin and ACE were distributed in vessel walls, mesenchymal cells, and in parts of surface epithelium. Angiotensinogen was also detected by RT-PCR. These findings demonstrated that Ang II receptors and RAS components were present in human colon, suggesting the possibility of its local regulation.
Immunocytochemical Demonstration of the Equilibrative Nucleoside Transporter rENT1 in Rat Sinoatrial Node
Authors: Musa H. , Dobrzynski H. , Berry Z. , Abidi F. , Cass C.E. , Young J.D. , Baldwin S.A. , Boyett M.R.
Published: April 2011

Adenosine exerts multiple receptor-mediated effects in the heart, including a negative chronotropic effect on the sinoatrial node. The aim of this study was to investigate the distribution of the equilibrative nucleoside transporter rENT1 in rat sinoatrial node and atrial muscle. Immunocytochemistry and/or immunoblotting revealed abundant expression of this protein in plasma membranes of sinoatrial node and in atrial and ventricular cells. Because rENT1-mediated transport is likely to regulate the local concentrations of adenosine in the sinoatrial node and other parts of the heart, it represents a potential pharmacological target that might be exploited to ameliorate ischemic damage during heart surgery.
Histochemical Localization of Alkaline Phosphatase Activity in Decalcified Bone and Cartilage
Authors: Miao Dengshun , Scutt Andrew
Published: April 2011

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.
Evidence of Oxidative Stress in Human Corneal Diseases
Authors: Buddi Rajeev , Lin Brian , Atilano Shari R. , Zorapapel Nadia C. , Kenney M. Cristina , Brown Donald J.
Published: April 2011

This study localized malondialdehyde (MDA, a toxic byproduct of lipid peroxidation), nitrotyrosine [NT, a cytotoxic byproduct of nitric oxide (NO)], and nitric oxide synthase isomers (NOS) in normal and diseased human corneas. Normal corneas ( n=11) and those with clinical and histopathological diagnoses of keratoconus ( n=26), bullous keratopathy ( n=17), and Fuchs' endothelial dystrophy ( n=12) were examined with antibodies specific for MDA, NT, eNOS (constitutive NOS), and iNOS (inducible NOS). Normal corneas showed little or no staining for MDA, NT, or iNOS, whereas eNOS was detected in the epithelium and endothelium. MDA was present in all disease groups, with each group displaying a distinct pattern of staining. NT was detected in all keratoconus and approximately one half of Fuchs' dystrophy corneas. iNOS and eNOS were evident in all the diseased corneas. Keratoconus corneas showed evidence of oxidative damage from cytotoxic byproducts generated by lipid peroxidation and the NO pathway. Bullous keratopathy corneas displayed byproducts of lipid peroxidation but not peroxynitrite (MDA but not NT). Conversely, Fuchs' dystrophy corneas displayed byproducts of peroxynitrite with little lipid peroxidation (NT » MDA). These data suggest that oxidative damage occurs within each group of diseased corneas. However, each disease exhibits a distinctive profile, with only keratoconus showing prominent staining for both nitrotyrosine and MDA. These results suggest that keratoconus corneas do not process reactive oxygen species in a normal manner, which may play a major role in the pathogenesis of this disease.
An Immunohistochemical Approach to Monitor the Prolactin-induced Activation of the JAK2/STAT5 Pathway in Pancreatic Islets of Langerhans
Authors: Brelje T. Clark , Svensson Annika M. , Stout Laurence E. , Bhagroo Nicholas V. , Sorenson Robert L.
Published: April 2011

This study examined whether an immunohistochemical method examining the subcellular localization of STAT5 could be used to characterize the activation of the JAK2/STAT5 pathway by prolactin (PRL) in intact cells or tissues. In the Ins-1 β-cell line, STAT5A and STAT5B were distributed almost equally in the cytoplasm and the nucleus in unstimulated cells. STAT5A was also detected along the border of cells and in the perinuclear region. After exposure to PRL, the redistribution from the cytoplasm to the nucleus was much higher for STAT5B compared to STAT5A. This translocation represented 12% of the STAT5A and 22% of the STAT5B originally located in the cytoplasm before stimulation. In isolated rat islets of Langerhans, PRL stimulated the nuclear translocation of both STAT5A and STAT5B only in β-cells. The expression of the PRL receptor only by β-cells was confirmed with a rabbit polyclonal antiserum raised against the rat PRL receptor. It was estimated that 4% of STAT5A and 9% of STAT5B originally located in the cytoplasm was translocated to the nucleus after stimulation. The presence of a functional JAK2/STAT5 signaling pathway in all islet cells was demonstrated by the nuclear translocation of STAT5B in all islet cells (i.e., α-, β-, and δ-cells) after stimulation with fetal calf serum. The nuclear translocation and tyrosine phosphorylation of STAT5B was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 hr, and prolonged activation after 4 hr. In contrast, the tyrosine phosphorylation of STAT5A was also biphasic but its nuclear translocation peaked within 30 min and was then reduced to a level slightly above that observed before PRL stimulation. This method is able to detect changes in STAT5 activation as small as 2% of the total cell content. These observations demonstrate the utility of this approach for studying the activation of STAT5 in a mixed population of cells within tissues or organs. In addition, the dose response for the nuclear translocation of STAT5B in normal β-cells was similar to those for changes in proliferation and insulin secretion in isolated rat islets. Therefore, the subcellular localization can be used to monitor the activation of STAT5 and it may be a key event in the upregulation of the pancreatic islets of Langerhans during pregnancy.
Smooth Muscle Myosin Heavy Chain Isoform Distribution in the Swine Stomach
Authors: Parisi Jonathan A. , Eddinger Thomas J.
Published: April 2011

To evaluate the distribution of smooth muscle myosin heavy chain isoforms (SMB, with head insert), we examined frozen sections from the various regions of swine stomachs using isoform-specific antibodies. We previously reported variable SMB myosin heavy chain (MHC) expression in stomach cells that correlates with unloaded shortening velocities. This is consistent with the generalization of tonic fundic muscle having low expression and phasic antral muscle having high expression of the SMB MHC isoform. Using im-munohistochemistry (IHC), we show a progression of the SMB MHC from very low immunoreactivity in the fundus to very intense immunoreactivity in the antrum. In the body, the average level of SMB MHC immunoreactivity lies between that of the antrum and fundus. Intercellular heterogeneity was observed in all stomach regions to a similar extent. However, the intercellular range in SMB MHC immunoreactivity decreases from fundus to antrum. All stomach regions show isolated pockets or clusters of cells with similar SMB MHC immunoreactivity. There is a non-uniform intracellular immunoreactivity in SMB MHC, with many cells showing greater-intensity staining of SMB MHC in their cell peripheries. This information may prove useful in helping to elucidate possible unique physiological roles of SMB MHC.
Tenascin-C Is Highly Expressed in Respiratory Distress Syndrome and Bronchopulmonary Dysplasia
Authors: Kaarteenaho-Wiik Riitta , Kinnula Vuokko L. , Herva Riitta , Soini Ylermi , Pöllänen Raimo , Pääkkö Paavo
Published: April 2011

Tenascin-C is an extracellular matrix (ECM) glycoprotein expressed in human tissues during organogenesis and in fibrotic and neoplastic processes. We hypothesized that its expression would increase in human lung in neonatal disorders such as infant respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Tenascin-C expression was studied by immunohistochemistry (IHC) and mRNA in situ hybridization (ISH). The extent of tenascin-C immunoreactivity was scored as absent (0), low (+), moderate (++), strong (+++), or very strong (++++) separately in different types of pulmonary cells in controls (seven cases), RDS (19 cases), and BPD (12 cases). In controls, tenascin-C expression was low (+) underneath alveolar and bronchiolar epithelium, moderate (++) in intima of veins, and strong (+++) around chondrocytes. In RDS, tenascin-C expression was moderate (++) or strong (+++) underneath both bronchiolar and often detached alveolar epithelium underlying hyaline membranes in the walls of dilated alveoli. In particular, the patients with RDS who survived for 1 day or more had strong expression of tenascin-C within alveolar walls. In patients with BPD, tenascin-C was very strongly (++++) expressed in the remodeled fibrotic alveolar walls underneath regenerative epithelium. Increased expression of tenascin-C mRNA was seen below the alveolar and bronchiolar epithelia in RDS and BPD. The cells in these locations showed α-smooth muscle actin immunoreactivity, suggesting a myofibroblast phenotype. In conclusion, tenascin-C is highly expressed in the walls of alveoli and bronchioli in RDS and BPD, suggesting an association between the expression of this protein and the presence of these disorders.
Human Lipocalin-1, a Physiological Scavenger of Lipophilic Compounds, Is Produced by Corticotrophs of the Pituitary Gland
Authors: Wojnar Petra , Dirnhofer Stephan , Ladurner Peter , Berger Peter , Redl Bernhard
Published: April 2011

Lipocalin-1 (Lcn-1), a member of the lipocalin superfamily that binds a broad array of different chemical classes of lipophilic ligands, is believed to act as a physiological scavenger of potentially harmful lipophilic molecules. Thus far, it was thought to be produced exclusively by a number of exocrine glands and tissues, including lachrymal and lingual glands, prostate, secretory glands of the tracheobronchial tract, and sweat glands. Using Northern blotting analysis, we were able to demonstrate Lcn-1 expression by the human pituitary gland. Moreover, double immunolabeling with antibodies against Lcn-1 and pituitary gland hormones and detection with fluorophore-conjugated secondary antibodies revealed that Lcn-1 is specifically produced by corticotrophs, clearly indicating that its distribution is not restricted to exocrine tissues.
Comparative Analysis of an Improved Thioflavin-S Stain, Gallyas Silver Stain, and Immunohistochemistry for Neurofibrillary Tangle Demonstration on the Same Sections
Authors: Sun Anyang , Nguyen Xuan V. , Bing Guoying
Published: April 2011

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.
Stanniocalcin 1 (STC1) Protein and mRNA Are Developmentally Regulated During Embryonic Mouse Osteogenesis: the Potential of STC1 as an Autocrine/Paracrine Factor for Osteoblast Development and Bone Formation
Authors: Yoshiko Yuji , Aubin Jane E. , Maeda Norihiko
Published: April 2011

STC1, a mammalian homologue of stanniocalcin (STC) which plays a major role in calcium/phosphate homeostasis in fish, has been recently isolated. We have characterized the spatiotemporal distribution of STC1 mRNA and protein during mouse embryonic development generally and osteogenesis specifically. Northern blotting analysis of whole embryos showed that STC1 mRNA is highly and differentially expressed during embryogenesis. By in situ hybridization, STC1 mRNA was detected early in mesenchymal condensations and was then found to be highly expressed in perichondrial cells, periosteal cells, and then osteoblasts during endochondral bone formation. In bones forming by intramembranous ossification, STC1 mRNA was not detected until osteogenic cells appeared. The cellular distribution of STC1 protein closely corresponded to that of its mRNA, but the protein was also detected in hypertrophic chondrocytes. In the MC3T3-E1 osteogenic cell model, STC1 protein and mRNA were detectable throughout proliferation and differentiation stages but levels were relatively higher late during nodule formation/mineralization phases. For comparison, STC1 mRNA was also found in epithelial cells of both embryonic and adult intestine that had not previously been described among tissues responsive to calcium/phosphate transport. These results suggest that STC1 is expressed in a time- and cell-specific manner and may play an autocrine/paracrine role during osteoblast development and bone formation.
Distribution of Melatonin MT1 Receptor Immunoreactivity in Human Retina
Authors: Savaskan Egemen , Wirz-Justice Anna , Olivieri Gianfranco , Pache Mona , Kräuchi Kurt , Brydon Lena , Jockers Ralf , Müller-Spahn Franz , Meyer Peter
Published: April 2011

Melatonin is synthesized in the pineal gland and retina during the night. Retinal melatonin is believed to be involved in local cellular modulation and in regulation of light-induced entrainment of circadian rhythms. The present study provides the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT1) in human retina of aged subjects. Ganglion, amacrine, and photoreceptor cells expressed MT1. In addition, MT1 immunoreactivity was localized to cell processes in the inner plexiform layer and to central vessels of the retina, as well as to retinal vessels but not to ciliary or choroidal vessels. These results support a variety of cellular and vascular effects of melatonin in the human retina. Preliminary evidence from patients with Alzheimer's disease (AD) revealed increased MT1 immunoreactivity in ganglion and amacrine cells, as well as in vessels. In AD cases photoreceptor cells were degenerated and showed low MT1 expression.
Improvement of Non-radioactive In Situ Hybridization in Human Airway Tissues: Use of PCR-generated Templates for Synthesis of Probes and an Antibody Sandwich Technique for Detection of Hybridization
Authors: Divjak Maja , Glare Eric M. , Walters E. Haydn
Published: April 2011

We describe the use of non-traditional methods of probe synthesis and quantification and detection of hybridization that appreciably improved non-radioactive in situ hybridization (ISH) in human airway tissue. To avoid the problems of bacterial cloning, plasmid digestion, and probe hydrolysis, we synthesised complementary RNA probes (riboprobes) for ISH from PCR-generated DNA. DNA template was produced by nested PCR incorporation of T7 and SP6 RNA polymerase promoters. We then compared the efficiency of in vitro transcription from PCR-generated template with traditional plasmid template by quantifying the relative probe fluorescence in denaturing gels. Transcription with SP6 or T7 polymerase in either orientation produced TNF riboprobes from a single PCR-generated template more efficiently than from plasmid, providing there were no primer hairpin loops. Fluorescence quantification enabled equal amounts of probe label to be used in ISH, eliminating signals from the sense probe and demonstrating that probes transcribed from PCR templates were as sensitive as hydrolyzed probe transcribed from plasmid. Detection of ISH by a conventional anti-hapten, alkaline phosphatase-based technique was found to cause tissue damage due to extended substrate incubation at high pH. We therefore developed a four-layer, avidin-biotin-peroxidase technique that afforded greater sensitivity, allowing brief substrate incubation and resulting in structural preservation of tissue.
Triple Immunofluorescence Staining with Antibodies Raised in the Same Species to Study the Complex Innervation Pattern of Intrapulmonary Chemoreceptors
Authors: Brouns Inge , Van Nassauw Luc , Van Genechten Jeroen , Majewski Mariusz , Scheuermann Dietrich W. , Timmermans Jean-Pierre , Adriaensen Dirk
Published: April 2011

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.
Apolipoprotein B Is Synthesized in Selected Human Non-hepatic Cell Lines But Not Processed into Mature Lipoprotein
Authors: Dixon Joseph L. , Biddle Jason , Lo Chun-min , Stoops J. Daniel , Sakata Hao Li Nobuhiro , Phillips Thomas E.
Published: April 2011

We studied apolipoprotein B100 (apoB) metabolism in a series of non-hepatic cell lines (HT29 colon adenocarcinoma, HeLa cervical epithelioid carcinoma, and 1321N1J astrocytoma human cell lines) and in the human hepatoma cell line HepG2. ApoB mRNA was detected by reverse transcription polymerase chain reaction in each non-hepatic cell line. ApoB was detected in HepG2 cells by immunoprecipitation, Western blotting, and immunocytochemistry using a polyclonal anti-human low-density lipoprotein (LDL) antibody, an anti-human apoB peptide antibody, and several monoclonal anti-apoB antibodies. ApoB was identified in the three non-hepatic cell lines by each method using the anti-apoB peptide and monoclonal antibodies, but not with the anti-LDL antibody. Im-munocytochemistry indicated that epitopes of apoB were evident throughout the endoplasmic reticulum, and gel mobility of newly labeled apoB and immunoblot with anti-ubiquitin showed that apoB was highly ubiquinated in non-hepatic cells. The observations that apoB is synthesized in non-hepatic cell lines but never recognized by the anti-LDL antibody suggests that apoB is not processed into a nascent lipoprotein in these cells. Immunocytochemical localization of apoB epitopes at many locations throughout non-hepatic cells raises the exciting possibility that apoB can be used for other purposes in these cells.
Immunolocalization of Surfactant Protein-D (SP-D) in Human Fetal, Newborn, and Adult Tissues
Authors: Stahlman Mildred T. , Gray Mary E. , Hull William M. , Whitsett Jeffrey A.
Published: April 2011

Immunoreactive surfactant protein-D (SP-D) was assessed in human fetal, newborn, and adult tissues. In the fetal lung, SP-D was detected on airway surfaces by 10 weeks' gestation, staining increasing in the distal airways, decreasing in the proximal conducting airways with advancing gestation. In lungs from near-term infants and adults, SP-D was detected in Type II cells, serous cells of tracheobronchial glands, and subsets of cells lining peripheral airways. Immunostaining was decreased or absent in areas of lungs of neonates after injury to Type II cells, infection, or hemorrhage and was decreased in collapsed or unseptated airways from older infants with bronchopulmonary dysplasia. SP-D was also detected in many organs at all ages. SP-D was readily detected in epithelial cells and luminal material in lacrimal glands, salivary glands, pancreas, bile ducts, renal tubules, esophageal muscle and glands, parietal cells of the stomach, crypts of Lieberkuhn, sebaceous and eccrine sweat glands, Von Ebner's glands, endocervical glands, seminal vesicles, adrenal cortex, myocardium, and anterior pituitary gland. SP-D is a widely distributed member of the “collectin” family of polypeptides secreted onto luminal surfaces by epithelial cells lining ducts of many organs, where it likely plays a role in innate host defense.
New Insights into Muscle Fiber Types in the Pig
Authors: Lefaucheur Louis , Ecolan Patrick , Plantard Lydia , Gueguen Naïg
Published: April 2011

The accurate classification of skeletal muscle fiber types according to myosin heavy chain (MyHC) polymorphism remains a difficult task in the pig. Combined myofibrillar ATPase and metabolic enzyme histochemistry, in situ hybridization, and immunocytochemistry were performed on serial transverse sections of pig longissimus (L) and rhomboideus (R) muscles at 100 kg body weight to give a new insight into muscle fiber typing in the pig. Several monoclonal antibodies (MAbs) either specific for a single MyHC (I, IIa, or IIb) or of multiple MyHCs (IIa + IIx or I + IIx + IIb) were used. No monospecific IIx antibody was available for the pig. All three adult Type II isoforms were expressed in the white L muscle, whereas no IIb was observed in the red R muscle, which was confirmed using RNase protection analysis. In most fibers, the distribution of the transcripts closely matched that of the corresponding proteins. When observed, co-expression of MyHCs mostly occured for IIx and IIb in L muscle, and was more common at the protein (11.5%) than at the mRNA (2.2%) level. A minor proportion of myofibers showed a mismatch between MyHC mRNA and protein. According to the type grouping distribution of myofibers encountered in pig muscle, MyHC isoform expression followed the rank order of I→Iia→Iix→IIb from the center to the periphery of the islets, concomitantly with a decrease in oxidative metabolism and an increase in fiber size. The developmental origin and functional significance of the type grouping distribution are discussed.
From Pixels to Picograms
Authors: Hardie David C. , Gregory T. Ryan , Hebert Paul D.N.
Published: April 2011

The study of genome size variation is important from a number of practical and theoretical perspectives. For example, the long-standing “C-value enigma” relating to the more than 200,000-fold range in eukaryotic genome sizes is best studied from a broad comparative standpoint. Genome size data are also required in detailed analyses of genome structure and evolution. The choice of future genome sequencing projects will be dependent on knowledge regarding the sizes of genomes to be sequenced, and so on. To date, genome size data have been acquired primarily by Feulgen microdensitometry or flow cytometry. Each has several advantages but also important limitations. In this review, we provide a practical guide to the new technique of Feulgen image analysis densitometry. The review is designed for those interested in genome size measurements but not extensively experienced in histochemistry, densitometry, or microscopy. Therefore, relevant historical and technical background information is included. For easy reference, we provide recipes for required reagents, guidelines for cell staining, and a checklist of steps for successful image analysis. We hope that the accuracy, rapidity, and cost-effectiveness of Feulgen image analysis demonstrated here will stimulate further surveys of genome sizes in a variety of taxa.
Expression of the Novel Epithelial Ca2+ Channel ECaC1 in Rat Pancreatic Islets
Authors: Janssen Susan W.J. , Hoenderop Joost G.J. , Hermus Ad R.M.M. , Sweep Fred C.G.J. , Martens Gerard J.M. , Bindels René J.M.
Published: April 2011

The epithelial Ca2+ channel, ECaC1, is primarily expressed in the apical membrane of vitamin D-responsive tissues. This study characterizes for the first time the presence of this novel channel in pancreatic tissue by reverse transcriptase-polymerase chain reaction and immunohistochemistry. In addition, the expression of ECaC1 was investigated in an animal model for Type 2 diabetes mellitus, the Zucker diabetic fatty (ZDF) rat. Identical staining patterns for ECaC1 and insulin were observed, whereas no co-localization of ECaC1 with glucagon was found. ECaC1, insulin, and prohormone convertase 1 (a neuroendocrine endoprotease expressed in secretory granules) showed a similar punctate staining. ECaC1 co-localized with the Ca2+ binding protein calbindin-D28K in the β-cells. Furthermore, in contrast to wild-type rats, in ZDF rats aging led to a progressive decrease in both insulin and ECaC1 staining. Plasma 1,25-dihydroxyvitamin D3 levels were similar in both control and ZDF rats and decreased with aging. Taken together, our findings indicate that this novel Ca2+ channel may play a role in the regulation of endocrine Ca2+ homeostasis.
Natriuretic Peptide System Gene Expression in Human Coronary Arteries
Authors: Casco Victor H. , Veinot John P. , de Bold Mercedes L. Kuroski , Masters Roy G. , Stevenson Michelle M. , de Bold Adolfo J.
Published: April 2011

The natriuretic peptides (NPs) ANF, BNP, and CNP have potent anti-proliferative and anti-migratory effects on vascular smooth muscle cells (SMCs). These properties make NPs relevant to the study of human coronary atherosclerosis because vascular cell proliferation and migration are central to the pathophysiology of atherosclerosis. However, the existence and cytological distribution of NPs and their receptors in human coronary arteries remain undetermined. This has hampered the development of hypotheses regarding the possible role of NPs in human coronary disease. We determined the pattern of expression of NPs and their receptors (NPRs) in human coronary arteries with atherosclerotic lesions classified by standard histopathological criteria as fatty streak/early atherosclerotic lesions, intermediate plaques, or advanced lesions. The investigation was carried out using a combination of immunocytochemistry (ICC), in situ hybridization (ISH), and semiquantitative polymerase chain reaction (PCR). Both by ICC and ISH, ANF was found in the intimal and medial layers of all lesions. BNP was highly expressed in advanced lesions where it was particularly evident by a strong ISH signal but weak ICC staining. CNP was demonstrable in all types of lesions, giving a strong signal by ISH and ICC. This peptide was particularly demonstrable in the endothelium, as well as in the SMCs of the intima, media, and vasa vasorum of the adventitia and in macrophages. By ISH, NPR-A was not detectable in any of the lesions but both NPR-B and NPR-C were found in the intimal and the inner medial layers. By RT-PCR, mRNA levels of all NPs tended to be increased in macroscopically diseased arteries, but only the values for BNP were significantly so. No significant changes in NPR mRNA levels were detected by PCR. In general, the signal intensity given by the NPs and their receptors by ICC or ISH appeared dependent on the type of lesion, being strongest in intermediate plaques and decreasing with increasing severity of the lesion. This study constitutes the first demonstration of NPs and NPR mRNAs in human coronary arteries and supports the existence of an autocrine/paracrine NP system that is actively modulated during the progression of atherosclerotic coronary disease. This suggests that the coronary NP system is involved in the pathobiology of intimal plaque formation in humans and may be involved in vascular remodeling.
The Murine mCLCA3 (Alias gob-5) Protein Is Located in the Mucin Granule Membranes of Intestinal, Respiratory, and Uterine Goblet Cells
Authors: Leverkoehne Ina , Gruber Achim D.
Published: April 2011

The putative anion channel mCLCA3 (alias gob-5) is the third murine member of the recently discovered family of calcium-activated chloride channels (CLCA family). Preliminary data suggest that mCLCA3 may play a significant role in diseases with secretory dysfunctions, including asthma and cystic fibrosis. In this study, the mCLCA3 protein was characterized biochemically and its cellular and subcellular distribution pattern was established in normal murine tissues. Polyclonal rabbit antibodies were generated and affinity-immunopurified using synthetic oligopeptides corresponding to the extracellular amino terminus of the mCLCA3 polypeptide. After in vitro translation and glycosylation, protein-ase K protection assay, and heterologous expression in COS-7 or HEK 293 cells, SDS-PAGE and immunoblotting revealed a protein structure similar to that of previously characterized CLCA proteins. A systematic light, confocal laser scanning, and transmission electron microscopic immunolocalization study, including virtually all murine tissues, identified the mCLCA3 protein exclusively associated with mucin granule membranes of gastrointestinal, respiratory, and uterine goblet cells and other mucin-producing cells. The results suggest that mCLCA3 may be involved in the synthesis, condensation, or secretion of mucins.
Effects of Infantile/Prepubertal Chronic Estrogen Treatment and Chemical Sympathectomy with Guanethidine on Developing Cholinergic Nerves of the Rat Uterus
Authors: Richeri Analía , Viettro Lorena , Chávez-Genaro Rebeca , Burnstock Geoffrey , Cowen Timothy , Brauer M. Mónica
Published: April 2011

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.
Detection of Pyruvate Dehydrogenase E1α-subunit Deficiencies in Females by Immunohistochemical Demonstration of Mosaicism in Cultured Fibroblasts
Authors: Lib Margarita Y. , Brown Ruth M. , Brown Garry K. , Marusich Michael F. , Capaldi Roderick A.
Published: April 2011

Deficiency of the E1α-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1α-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1α deficiency. We also show that anti-E1α and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2–5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1α-subunit deficiency as a precursor to mutation analysis.
Estimation of the Total Number of Cholinergic Neurons Containing Estrogen Receptor-α in the Rat Basal Forebrain
Authors: Miettinen Riitta A. , Kalesnykas Giedrius , Koivisto Esa H.
Published: April 2011

This study was undertaken to estimate the total number of cholinergic cells and the percentage of cholinergic cells that contain estrogen receptor-α (ERα) in the rat basal forebrain. Double immunostaining for choline acetyltransferase (ChAT) and ERα was carried out on 50-μm-thick free-floating sections. Because routine mounting method causes considerable flattening of the sections, we embedded immunostained sections in Durcupan, an epoxy resin known to cause virtually no shrinkage. When this procedure was used the section thickness was well preserved, individual cells could be clearly identified, and subcellular localization of ERα immunoreactivity was easy to verify. Cell counting in these sections revealed that the rat basal forebrain contains 26,390 ± 1097 (mean ± SEM) cholinergic neurons. This comprises 9674 ± 504 in the medial septum-vertical diagonal band of Broca, 9403 ± 484 in the horizontal diagonal band of Broca, and 7312 ± 281 in the nucleus basalis. In these nuclei, 60%, 46%, and 14% of the cholinergic neurons were colocalized with ERα, respectively. We believe that our results are an improvement on existing data because of the better distinction of individual neurons that the Durcupan embedding method brings.
Immunocytochemical Localization of Prohormone Convertases PC1 and PC2 in the Mouse Thyroid Gland and Respiratory Tract
Authors: Kurabuchi Shingo , Tanaka Shigeyasu
Published: April 2011

We examined immunocytochemical localization of the prohormone convertases, PC1 and PC2, in the thyroid gland and respiratory tract of the adult mouse using the indirect enzyme- and immunogold-labeled antibody methods for light and electron microscopy, respectively. In the thyroid gland, PC1- and/or PC2-immunoreactive cells were cuboidal, scattered in the follicular epithelium and in the interfollicular spaces. When serial sections were immunostained with anti-calcitonin, anti-PC1, anti-calcitonin-gene-related-peptide (CGRP), and anti-PC2 sera, respectively, localization of both PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells. In the respiratory tract, only PC1 immunoreactivity was observed in the basal granulated neuroendocrine cells, which were scattered in the tracheal epithelium. Consecutive sections immunostained with anti-PC1 and anti-CGRP sera showed that a subpopulation of these PC1-immunoreactive cells contained CGRP. Double immunogold electron microscopy of the thyroid parafollicular cells revealed that calcitonin- and/or CGRP-immunopositive secretory granules were also labeled with both PC1 and PC2. These findings suggest that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.
Cellular Pathway of Plasmids Vectorized by Cholesterol-based Cationic Liposomes
Authors: Briane Dominique , Lesage Denis , Cao An , Coudert Robert , Lievre Nicole , Salzmann Jean Loup , Taillandier Eliane
Published: April 2011

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3β( N-( N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3β( N-( N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.
DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Principle: Heating Under the Influence of pH
Authors: Shi Shan-Rong , Cote Richard J. , Wu Lin , Liu Cheng , Datar Ram , Shi Yan , Liu Dongxin , Lim Hyoeun , Taylor Clive R.
Published: April 2011

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6–9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6–12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.
Ghrelin Expression in Fetal, Infant, and Adult Human Lung
Authors: Volante Marco , Fulcheri Ezio , Allìa Elena , Cerrato Milena , Pucci Angela , Papotti Mauro
Published: April 2011

Ghrelin is a recently identified hormone with potent growth hormone (GH)-releasing activity. It is produced by rat and human gastric endocrine cells and by the pituitary, hypothalamus, placenta, and by gastroenteropancreatic tumors. No evidence of ghrelin production by foregut-derived organs other than stomach has been provided to date. The aim of the present study was to investigate ghrelin expression by human fetal (20 cases), infant (13 cases), and adult (seven cases) lungs by immunohistochemistry, in situ hybridization, and RT-PCR. Expression of the GH secretagogue receptor, the endogenous receptor for ghrelin, was also investigated by RT-PCR. Ghrelin protein was found in the endocrine cells of the fetal lung in decreasing amounts from embryonic to late fetal periods. Its expression was maintained in newborns and children under 2 years but was virtually absent in older individuals. Scattered positive cells were also found in the trachea and the esophagus. Ghrelin mRNA was detected in adult lung by the more sensitive RT-PCR technique. GHS receptor mRNA was detected in nine cases of infant and adult lungs, possibly indicating the existence of local autocrine circuits. We conclude that the fetal lung is an additional source of circulating ghrelin, whose functions at the respiratory tract level remain to be clarified.