bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Neural Control of Myogenic Regulatory Factor Genes During Regeneration of Mouse Soleus
Authors: Launay Thierry , Armand Anne-Sophie , Charbonnier Frédéric , Mira Jean-Claude , Donsez Evelyne , Gallien Claude L. , Chanoine Christophe
Published: April 2011

Given the importance of the myogenic regulatory factors (MRFs) for myoblast differentiation during development, the aims of this work were to clarify the spatial and temporal expression pattern of the four MRF mRNAs during soleus regeneration in mouse after cardiotoxin injury, using in situ hybridization, and to investigate the influence of innervation on the expression of each MRF during a complete degeneration/regeneration process. For this, we performed cardiotoxin injury-induced regeneration experiments on denervated soleus muscle. Myf-5, MyoD, and MRF4 mRNAs were detected in satellite cell-derived myoblasts in the first stages of muscle regeneration analyzed (2–3 days P-I). The Myf-5 transcript level dramatically decreased in young multinucleated myotubes, whereas MyoD and MRF4 transcripts were expressed persistently throughout the regeneration process. Myogenin mRNA was transiently expressed in forming myotubes. These results are discussed with regard to the potential relationships between MyoD and MRF4 in the satellite cell differentiation pathway. Muscle denervation precociously (at 8 days P-I) upregulated both the Myf-5 and the MRF4 mRNA levels, whereas the increase of both MyoD and myogenin mRNA levels was observed later, in the late stages of regeneration (30 days P-I). This significant accumulation of each differentially upregulated MRF during soleus regeneration after denervation suggests that each myogenic factor might have a distinct role in the regulatory control of muscle gene expression. This role is discussed in relation to the expression of the nerve-regulated genes, such as the nAChR subunit gene family.
Comparison of Apoptosis and Terminal Differentiation: The Mammalian Aging Process
Authors: Gagna C.E. , Kuo H.-R. , Florea E. , Shami W. , Taormina R. , Vaswani N. , Gupta M. , Vijh R. , Lambert W.C.
Published: April 2011

Apoptosis is the ordered chain of events that lead to cell destruction. Terminal differentiation (denucleation) is the process in which cells lose their nuclei but remain functional. Our group examined cell death in three tissues using two different fixatives and a postfixation procedure, involving young (5 months) and old (2 years) guinea pigs. The data reveal that B-DNA and Z-DNA content decreases, whereas single-stranded (ss-) DNA increases, in older tissues undergoing apoptosis (skin and cornea) and terminal differentiation (ocular lens). We speculate that some of the factors that contribute to the aging process might also be responsible for the enhanced amount of damaged DNA in older tissues undergoing cell death.
Isolation of Mature Spinal Motor Neurons and Single-cell Analysis Using the Comet Assay of Early Low-level DNA Damage Induced In Vitro and In Vivo
Authors: Liu Zhiping , Martin Lee J.
Published: April 2011

We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in α-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration. (J Histochem Cytochem 49:957–972, 2001)
SLC26A2 (Diastrophic Dysplasia Sulfate Transporter) is Expressed in Developing and Mature Cartilage But Also in Other Tissues and Cell Types
Authors: Haila Siru , Hästbacka Johanna , Böhling Tom , Karjalainen–Lindsberg Marja-Liisa , Kere Juha , Saarialho–Kere Ulpu
Published: April 2011

Mutated alleles of the SLC26A2 (diastrophic dysplasia sulfate transporter or DTDST) gene cause each of the four recessive chondrodysplasias, i.e., diastrophic dysplasia (DTD), multiple epiphyseal dysplasia (MED), atelosteogenesis Type II (AO2), and achondrogenesis Type IB (ACG1B). SLC26A2 acts as an Na+-independent sulfate/chloride antiporter and belongs to the SLC26 anion transporter gene family, currently consisting of six homologous human members. Although Northern analysis has indicated some expression in all tissues studied, the only tissue known to be affected by SLC26A2 mutations is cartilage. Abundant SLC26A2 expression has previously been detected in normal human colon by in situ hybridization. We have used in situ hybridization and immunohistochemistry to examine multiple normal tissues for the expression of human SLC26A2. As expected, a strong signal for SLC26A2 mRNA and protein immunostaining were detected in developing fetal hyaline cartilage, while bronchial cartilage showed mRNA expression in adult tissues. SLC26A2 expression could also be detected in eccrine sweat glands, in bronchial glands, and in placental villi. In addition, immunoreactivity for the SLC26A2 protein was observed in exocrine pancreas. Our results suggest a more limited expression pattern for SLC26A2 than that found by Northern analysis. However, SLC26A2 expression is also detected in tissues not affected in chondrodysplasias caused by SLC26A2 mutations. (J Histochem Cytochem 49:973–982, 2001)
Computerized Image Analysis as a Tool to Quantify Infiltrating Leukocytes
Authors: Johansson Anna C. , Visse Edward , Widegren Bengt , Sjögren Hans-Olov , Siesjö Peter
Published: April 2011

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a ×100 or a ×12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-γ (IFN-γ) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software. (J Histochem Cytochem 49:1073–1079, 2001)
Immunohistochemical Localization of Pax2 and Associated Proteins in the Developing Kidney of Mice with Renal Hypoplasia
Authors: Lozanoff Scott , Johnston Jayne , Ma Wenbin , Saux Claude Jourdan–Le
Published: April 2011

Pax2 has been identified as a key regulatory protein associated with renal developmental malformations. The purpose of this study was to determine whether Pax2 protein expression, and that of other proteins important for normal renal development, is abnormally distributed in the prenatal kidney of the Brachyrrhine ( Br) mouse that displays heritable renal hypoplasia. Embryonic 3H1 +/+ and Br/Br mice were collected between E11.0 and E18.0. Routine light microscopy and immunohistochemical analysis using antibodies to Pax2, E-cadherin, fibronectin, laminin, and Type IV collagen were applied to sequential tissue sections. E-cadherin stained consistently in the renal tubules of both normal and mutant animals. Whereas the initial expression of Pax2 corresponded between normal and mutant kidneys, it became progressively limited to the nephrogenic zone in +/+ animals, while distributing erratically in the Br/Br kidney. Fibronectin was not expressed in the normal nephrogenic zone but remained abundantly distributed throughout the Br/Br kidney. Luminin and Type IV collagen staining revealed a deficiency in renal vasculature formation in Br/Br kidneys. Results suggest that initial morphological differentiation occurs normally in the Br kidney but that subsequent nephric formation is associated with abnormal distribution of Pax2 and ECM proteins. (J Histochem Cytochem 49:1081–1097, 2001)
High-resolution Immunocytochemistry of Noncollagenous Matrix Proteins in Rat Mandibles Processed with Microwave Irradiation
Authors: Arana–Chavez Victor E. , Nanci Antonio
Published: April 2011

The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at temperatures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2, for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR White resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention. (J Histochem Cytochem 49:1099–1109, 2001)
Localization of Cytosolic NADP-dependent Isocitrate Dehydrogenase in the Peroxisomes of Rat Liver Cells
Authors: Yoshihara Takashi , Hamamoto Tatsuhiko , Munakata Ryo , Tajiri Ryosuke , Ohsumi Mariko , Yokota Sadaki
Published: April 2011

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123–1131, 2001)
Prolyl 4-hydroxylase Isoenzymes I and II Have Different Expression Patterns in Several Human Tissues
Authors: Nissi Ritva , Autio–Harmainen Helena , Marttila Pia , Sormunen Raija , Kivirikko Kari I.
Published: April 2011

Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens. We have previously reported that the recently identified Type II isoenzyme is its main form in chondrocytes and possibly in capillary endothelial cells, while Type I is the main form in many other cell types. We report here that the Type II isoenzyme is clearly the main form in capillary endothelial cells and also in cultured umbilical vein endothelial cells, whereas no Type I isoenzyme could be detected in these cells by immunostaining or Western blotting. The Type II isoenzyme was also the main form in cells of the developing glomeruli in the fetal kidney and tubular structures of collecting duct caliber in both fetal and adult kidney, in occasional sinusoidal structures and epithelia of the bile ducts in the liver, and in some cells of the decidual membrane that probably represented invasive cytotrophoblasts in the placenta. Osteoblasts in a fetal calvaria, i.e., a bone developing by intramembranous ossification, stained strongly for both types of isoenzyme. The Type I isoenzyme was the main form in undifferentiated interstitial mesenchymal cells of the developing kidney, for example, and in fibroblasts and fibroblastic cells in many tissues. Skeletal myocytes and smooth muscle cells appeared to have the Type I isoenzyme as their only prolyl 4-hydroxylase form. Hepatocytes expressed small amounts of the Type I enzyme and very little if any Type II, the Type I expression being increased in malignant hepatocytes and cultured hepatoblastoma cells. The data suggest that the Type I isoenzyme is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas the Type II isoenzyme is expressed, in addition to chondrocytes and osteoblasts, by more differentiated cells, such as endothelial cells and cells of epithelial structures. (J Histochem Cytochem 49:1143–1153, 2001)
Simultaneous Detection of RNA and Protein by In Situ Hybridization and Immunological Staining
Authors: Nagaso Hideyuki , Murata Takehide , Day Noel , Yokoyama Kazunari K.
Published: April 2011

Proteinase K is widely used in methods for detection of transcripts in biological specimens by in situ hybridization (ISH). However, treatment with proteinase K hampers detection of RNA and protein simultaneously. We have developed a method for double staining of transcripts and proteins by ISH and IHC staining in imaginal discs and embryos of Drosophila. Instead of treatment with proteinase K, samples are treated with ethanol plus xylene and with acetone. Acetone renders cell membranes permeable to probes and antibodies without damaging tissue integrity, whereas treatment with proteinase K sometimes damages tissues. Treatment of samples with acetone allows hybridization of probe with transcripts in tissue. It is also effective for immunological staining of samples after ISH with a riboprobe. Thus, our method allows detection not only of transcripts but also of specific proteins in relatively intact single samples. (J Histochem Cytochem 49:1177–1182, 2001)
Stability of Nitroblue Tetrazolium-based Alkaline Phosphatase Substrates
Authors: Smejkal Gary B. , Kaul Charles A.
Published: April 2011

This report demonstrates the stability of NBT substrate after multiple exposures to alkaline phosphatase. Perhaps more important than the ability to reuse substrates, the report provides some insight into the mechanisms by which tetrazoliums are reduced and evidence for the formation of an intermediary product, i.e., a half-formazan that is reduced more rapidly. (J Histochem Cytochem 49:1189–1190, 2001)
HPV for Cervical Cancer Screening
Authors: Stoler Mark H.
Published: April 2011

Based on the best estimates of the prevalence of human Papillomavirus infection in the United States, the overall HPV prevalence in the target population is approximately 20%. The prevalence varies greatly with age, being as high as ∼50% in the third decade to less than 5% in the sixth. These data have implications for a discussion about the utility of human Papillomavirus testing as a screening procedure. (J Histochem Cytochem 49:1197–1198, 2001)
Changes in the Distribution of Anionic Constituents in Secretory Granules of Mouse Pancreatic Acinar Cells After Pilocarpine-induced Degranulation
Authors: Shoichetman Tanya , Skutelsky Ehud , Lew Sylvia , Hammel Ilan
Published: April 2011

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpinestimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (≈43.0 gold particles/μm2) compared to the 8-hr mature granules group (≈64.6 gold particles/μm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.
Reduced Expression of Activin A in Focal Lymphoid Agglomerates Within Nasal Polyps
Authors: Shoham Tsipi , Yaniv Eitan , Koren Rumelia , Gal Rivka , Parameswaran Reshmi , Kravitz Arkadi , Geron Henry , Markovitz Dana , Lantzki Marina , Zipori Dov
Published: April 2011

It has been previously reported that activin A, a homodimer of the βA inhibin subunit, is secreted by stromal cells from mouse bone marrow and causes apoptotic death of mouse plasmacytoma tumor cells. Recent in vitro studies have also implicated this cytokine in the suppression of normal B-cell lymphopoiesis. In this study we examined the occurrence of activin A in nasal polyp tissues that present a combination of epithelium, mesenchyme, and vascular endothelium, with frequent massive hemopoietic infiltration. Anti-βA-chain antibodies strongly stained epithelial mucous glands and some endothelial cells, and diffusely stained the polyp stroma. Normal adult conchae were similarly stained, whereas activin A was not detected prenatally by immunostaining of nasal tissues. Staining specificity was substantiated by ligand competition assays. Detailed examination of the inflammatory polyp infiltrate showed that activin A staining was reduced in sites of focal infiltration of B-lymphoid cells. It is therefore implied that local accumulation of a large number of B-cells is associated with relatively low activin A expression.
Expression of Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain, a Pyrin N-terminal Homology Domain-containing Protein, in Normal Human Tissues
Authors: Masumoto Junya , Taniguchi Shun'ichiro , Nakayama Jun , Shiohara Masaaki , Hidaka Eiko , Katsuyama Tsutomu , Murase Sumio , Sagara Junji
Published: April 2011

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a pyrin N-terminal homology domain (PYD)- and caspase recruitment domain (CARD)-containing a proapoptotic molecule. This molecule has also been identified as a target of methylation-induced silencing (TMS)-1. We cloned the ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody. In this study, we determined the binding site of the anti-ASC monoclonal antibody on ASC and analyzed the expression of ASC in normal human tissues. ASC expression was observed in anterior horn cells of the spinal cord, trophoblasts of the placental villi, tubule epithelium of the kidney, seminiferous tubules and Leydig cells of the testis, hepatocytes and interlobular bile ducts of the liver, squamous epithelial cells of the tonsil and skin, hair follicle, sebaceous and eccrine glands of the skin, and peripheral blood leukocytes. In the colon, ASC was detected in mature epithelial cells facing the luminal side rather than immature cells located deeper in the crypts. These observations indicate that high levels of ASC are abundantly expressed in epithelial cells and leukocytes, which are involved in host defense against external pathogens and in well-differentiated cells, the proliferation of which is regulated.
Color Subtractive–Computer-assisted Image Analysis for Quantification of Cutaneous Nerves in a Diabetic Mouse Model
Authors: Underwood Robert A. , Gibran Nicole S. , Muffley Lara A. , Usui Marcia L. , Olerud John E.
Published: April 2011

Immunohistochemistry (IHC) is a valuable tool for labeling structures in tissue samples. Quantification of immunolabeled structures using traditional approaches has proved to be difficult. Manual counts of IHC-stained structures are inherently biased, require multiple observers, and generate qualitative data. Stereological methods provide accurate quantification but are complex and labor-intensive when staining must be compared among large numbers of samples. In an effort to quickly, objectively, and reproducibly quantify cutaneous innervation in a large number of counterstained tissue sections, we developed a color subtractive–computer-assisted image analysis (CS–CAIA) system. To develop and test the CS–CAIA method, tissue sections of diabetic (db/db) mouse skin and their wild-type (db/–) littermates were stained by IHC for the neural marker PGP 9.5. The brown-red PGP 9.5 peroxidase stain was colorimetrically isolated through a scripted process of color background removal. The remaining stain was thresholded and binarized for computer determination of nerve profile counts (number of stained regions), area fraction (total area of nerve profiles per unit area of tissue), and area density (total number of nerve profiles per unit area of tissue). Using CS–CAIA, epidermal nerve profile counts, area fraction, and area density were significantly lower in db/db compared to db/– mice.
Location of Sialoglycoconjugates Containing the Siaα2–3Gal and Siaα2–6Gal Groups in the Rat Hippocampus and the Effect of Aging on Their Expression
Authors: Sato Yuji , Akimoto Yoshihiro , Kawakami Hayato , Hirano Hiroshi , Endo Tamao
Published: April 2011

The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Siaα2–3Gal, and Sambucus sieboldiana agglutinin, specific for Siaα2–6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Siaα2–3Gal and Siaα2–6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum moleculare. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Siaα2–3Gal and Siaα2–6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain.
Towards a Molecular Classification of Hematolymphoid Neoplasms: Where Are We Now? Where Are We Going?
Authors: Hsi Eric D.
Published: April 2011

Beginning with the discovery of the Philadelphia chromosome in 1960, our descriptions and definitions of hematological malignancies have been successively refined and revised to include morphological, immunophenotypic, and molecular genetic features. As hematopathologists, we now see beyond architectural and cytologic alterations and have, to some degree, become molecular morphologists. Indeed, our “view” of diseases now includes a rudimentary understanding of the molecular genetic abnormalities present in the cells we study under the microscope.
In Vivo Transduction of Central Neurons Using Recombinant Sindbis Virus
Authors: Furuta Takahiro , Tomioka Ryohei , Taki Kousuke , Nakamura Kouichi , Tamamaki Nobuaki , Kaneko Takeshi
Published: April 2011

A new recombinant virus which labeled the infected neurons in a Golgi stainlike fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (pal-GFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.
Novel Non-isotopic Method for the Localization of Receptors in Tissue Sections
Authors: Desnoyers Luc , Simonette Rebecca A. , Vandlen Richard L. , Fendly Brian M.
Published: April 2011

We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.
Immunohistochemistry of PrPsc Within Bovine Spongiform Encephalopathy Brain Samples with Graded Autolysis
Authors: Debeer Sabine O.S. , Baron Thierry G.M. , Bencsik Anna A.
Published: April 2011

Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis.
Characterization of a Polyclonal Antibody to Human Pituitary Tumor Transforming Gene 1 (PTTG1) Protein
Authors: Kakar Sham S. , Chen Leilei , Puri Rashmi , Flynn Shawn E. , Jennes Lothar
Published: April 2011

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His6 tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.
Apoptosis and Proliferation of Myoepithelial Cells in Atrophic Rat Submandibular Glands
Authors: Takahashi Shigeru , Nakamura Shiro , Shinzato Katsuhiro , Domon Takanori , Yamamoto Tsuneyuki , Wakita Minoru
Published: April 2011

This study was designed to determine whether apoptosis and proliferation of myoepithelial cells occur in atrophic rat submandibular glands. The excretory duct of the right submandibular gland was doubly ligated with metal clips. The atrophic right submandibular glands removed after 1–28 days of duct ligation were investigated using immunohistochemical double staining for actin as a marker for myoepithelial cells and proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells, double staining for actin immunohistochemistry, nick end-labeling (TUNEL) as a marker for apoptotic cells, and transmission electron microscopy (TEM). A few PCNA- and no TUNEL-positive myoepithelial cells were found in the control submandibular glands taken from animals with no operation. In the experimental glands, PCNA-positive myoepithelial cells were common 2 and 3 days after duct ligation and then decreased in number. TUNEL-positive myoepithelial cells appeared at 2 days and were observed most frequently at 5 days. Apoptotic myoepithelial cells were also identified by TEM. These observations suggest that both apoptosis and proliferation of myoepithelial cells occur, especially in the early phase of atrophy, in the rat submandibular gland.
Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)
Authors: Baschong Werner , Suetterlin Rosmarie , Laeng R. Hubert
Published: April 2011

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.
Functional Interaction of Caveolin-1 and eNOS in Myocardial Capillary Endothelium Revealed by Immunoelectron Microscopy
Authors: Reiner Michael , Bloch Wilhelm , Addicks Klaus
Published: April 2011

Immunogold labeling on samples of isolated perfused rat hearts embedded by an innovative low-temperature LR White procedure provided detailed insight into the interaction of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level. Separately, the localization of caveolin-1 and eNOS at caveolae under steady state conditions was visualized. A double-labeling experiment supported their close co-localization. Short-term bradykinin stimulation caused a detectable dissociation of eNOS from caveolin and its redistribution to different cell compartments, whereas caveolin itself remained stationary at caveolae. Morphometric analysis revealed that more than 80% of detectable eNOS was co-localized with caveolin-1 at caveolae under control conditions. After brief stimulation for 2 min with 10-7 M bradykinin, only 26% of the eNOS signals were associated with caveolin-1 and randomly distributed over the endothelial cells. After stimulation, eNOS was found at the plasmalemmal and intracellular membranes, freely in the cytoplasm, and at outer mitochondrial membranes.
Immunocytochemical Detection of Orexin A in Endocrine Cells of the Developing Mouse Gut
Authors: Miguel María J. Sánchez de , Burrell María A.
Published: April 2011

Orexins are novel neuropeptides that were originally localized in neurons of the hypothalamus and neuronal fibers of the brain. Recently orexin A and its receptor have also been reported in neurons and endocrine cells of the gastrointestinal tract. Because no studies have been done at the embryonic period, we studied the appearance and distribution of orexin A during the development of mouse gastrointestinal tract using immunocytochemical methods. Immunoreactivity to orexin A was detected in neuroendocrine cells of the pyloric region of the stomach at gestational Day 14 and 1 day after in the small intestine. The numbers of immunoreactive cells progressively increased through development until the adult pattern was reached. Staining of reverse-face sections demonstrated that orexin A and serotonin co-localized in some endocrine cells of the mouse stomach and small intestine. These findings suggest that orexin A may be relevant in the growth and maturation of the gastrointestinal tract during intrauterine life.
Morphological Characteristics of the Microvasculature in Healing Myocardial Infarcts
Authors: Ren Guofeng , Michael Lloyd H. , Entman Mark L. , Frangogiannis Nikolaos G.
Published: April 2011

Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor “mother vessels” and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 ± 6.94% after 28 days of reperfusion vs 30.17 ± 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair.
Expression of the Gene Encoding the LIM Protein CRP2: A Developmental Profile
Authors: Henderson James R. , Brown Doris , Richardson James A. , Olson Eric N. , Beckerle Mary C.
Published: April 2011

Members of the cysteine-rich protein (CRP) family are evolutionarily conserved Lin-11, Isl-1, Mac-3 (LIM) domain proteins that have been implicated in cell differentiation. Here we describe the expression pattern of the CRP family member CRP2 in mouse. Unlike other CRP family members, which are expressed primarily in muscle, CRP2 is more broadly expressed. In addition to expression in vascular smooth muscle cells, we also detect CRP2-specific transcripts in mesenchymal derivatives and several epithelia.
Myosin Heavy Chain Composition of Muscle Spindles in Human Biceps Brachii
Authors: Liu Jing-Xia , Eriksson Per-Olof , Thornell Lars-Eric , Pedrosa-Domellöf Fatima
Published: April 2011

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag1 fibers contained slow tonic MyHC along their entire fiber length and MyHCI, α-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag2 fibers contained MyHC slow tonic, I, α-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag1, bag2 and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.
Fluid Dynamics of the Excretory Flow of Zymogenic and Mucin Contents in Rat Gastric Gland Processed by High-pressure Freezing/Freeze Substitution
Authors: Sawaguchi Akira , Ishihara Kazuhiko , Kawano Jun-ichi , Oinuma Tsutomu , Hotta Kyoko , Suganuma Tatsuo
Published: April 2011

The high-pressure freezing/freeze substitution technique followed by Lowi-cryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO4-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.
Fluorescent Histochemical Techniques for Analysis of Intracellular Signaling
Authors: Oksvold Morten P. , Skarpen Ellen , Widerberg Jannicke , Huitfeldt Henrik S.
Published: April 2011

Intracellular signaling relies on the orchestrated cooperation of signaling proteins and modules, their intracellular localization, and membrane trafficking. Recently, a repertoire of fluorescence-based techniques, which significantly increases our potential for detailed studies of the involved mechanisms, has been introduced. Microscopic techniques with increased resolution have been combined with improved techniques for detection of signaling proteins. Transfections of fluorescently tagged proteins have allowed in vivo microscopy of their trafficking and interactions with other proteins and intracellular structures. We present an overview of general signaling principles and a description of techniques based on fluorescent microscopy suited for studies of signaling mechanisms.
Heterogeneous Expression of Ca2+ Handling Proteins in Rabbit Sinoatrial Node
Authors: Musa Hanny , Lei Ming , Honjo Hauro , Jones Sandra A. , Dobrzynski Halina , Lancaster Mathew K. , Takagishi Yoshiko , Henderson Zaineb , Kodama Itsuo , Boyett Mark R.
Published: April 2011

We investigated the densities of the L-type Ca2+ current, iCa,L, and various Ca2+ handling proteins in rabbit sinoatrial (SA) node. The density of iCa,L, recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of iCa,L was significantly ( p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca2+ channel, Na+-Ca2+ exchanger, sarcoplasmic reticulum Ca2+ release channel (RYR2), and sarcoplasmic reticulum Ca2+ pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly ( p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na+-K+ pump was similar in peripheral and central cells. We conclude that Ca2+ handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).
Inhibitor Profiles of Alkaline Phosphatases in Bovine Preattachment Embryos and Adult Tissues
Authors: McDougall K. , Plumb C. , King W.A. , Hahnel A.
Published: April 2011

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or −3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.