bluuebunny/crossref_metadata_2025 huggingface dataset:
Simple and Rapid Methods for SEM Observation and TEM Immunolabeling of Rubber Particles
Authors: Singh Adya P. , Wi Seung Gon , Kang Hunseung , Chung Gap Chae , Kim Yoon Soo
Published: April 2011

We developed a method involving air-drying of a rubber suspension after fixation in glutaraldehyde-tannic acid and postfixation in osmium tetroxide for SEM observation. For TEM immunolabeling the suspension was air-dried after osmium-only fixation. Whereas conventional methods failed to satisfactorily stabilize rubber particles, the methods described here proved successful in preserving their integrity.
Widespread Expression of Arginase I in Mouse Tissues: Biochemical and Physiological Implications
Authors: Yu Hong , Yoo Paul K. , Aguirre Claudia C. , Tsoa Rosemarie W. , Kern Rita M. , Grody Wayne W. , Cederbaum Stephen D. , Iyer Ramaswamy K.
Published: April 2011

Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells.
Applicability of Different Antibodies for the Immunohistochemical Localization of CFTR In Respiratory and Intestinal Tissues of Human and Murine Origin
Authors: Doucet Laurent , Mendes Filipa , Montier Tristan , Delépine Pascal , Penque Deborah , Férec Claude , Amaral Margarida D.
Published: April 2011

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl−) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies's sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.
Triple Immunofluorolabeling with Two Rabbit Polyclonal Antibodies and a Mouse Monoclonal Antibody Allowing Three-dimensional Analysis of Cotton Wool Plaques in Alzheimer Disease
Authors: Uchihara Toshiki , Nakamura Ayako , Nakayama Hiroshi , Arima Kunimasa , Ishizuka Norio , Mori Hiroshi , Mizushima Setsuo
Published: April 2011

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Aβ42 or anti-Aβ40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Aβ40 or anti-Ap42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 μm were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Aβ40 was localized to core-like structures, mainly in layers I–III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I–VI, were labeled with anti-Aβ42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Aβ deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.
Differential Expression of Equine Myosin Heavy-chain mRNA and Protein Isoforms in a Limb Muscle
Authors: Eizema Karin , van den Burg Maarten , Kiri Arpna , Dingboom Elizabeth G. , van Oudheusden Hans , Goldspink Geoffrey , Weijs Wim A.
Published: April 2011

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.
Immunolocalization of Sperm Protein 17 in Human Testis and Ejaculated Spermatozoa
Authors: Grizzi Fabio , Chiriva–Internati, Maurizio , Franceschini Barbara , Hermonat Paul L. , Soda Giuseppe , Lim Seah H. , Dioguardi Nicola
Published: April 2011

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.
Extended Chromatin and DNA Fibers from Active Plant Nuclei for High-resolution FISH
Authors: Lavania U.C. , Yamamoto M. , Mukai Y.
Published: April 2011

The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G1 nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency.
Distribution of the Serine Protease HtrA1 in Normal Human Tissues
Authors: De Luca Antonio , De Falco Maria , Severino Anna , Campioni Mara , Santini Daniele , Baldi Feliciano , Paggi Marco G. , Baldi Alfonso
Published: April 2011

The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types.
p16INK4A Immunohistochemical Overexpression in Premalignant and Malignant Oral Lesions Infected with Human Papillomavirus
Authors: Gabrielli Fregonesi Paula Andrea , Teresa Debora Barreto , Duarte Roberta Aparecida , Neto Carlos Benatti , de Oliveira Maria Rita Brancini , Soares Christiane Pienna
Published: April 2011

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA–ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.
Ultrastructural Orientation of Laminin 5 in the Epidermal Basement Membrane: an Updated Model for Basement Membrane Organization
Authors: McMillan James R. , Akiyama Masashi , Shimizu Hiroshi
Published: April 2011

Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the α3-chain first globular domain) that was measured at 35.40 ± 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the β3-chain globular domain IV) that measured 45.20 ± 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 ± 6.28 nm from the PM. The laminin 5 γ2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 ± 1.65 nm from the keratinocyte PM. Taken together with previous γ2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27° from the horizontal lamina lucida (LL)/LD border and propose that the γ2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.
Expression of the Integrin Subunit α8 in Murine Lung Development
Authors: Wagner Teresa E. , Frevert Charles W. , Herzog Erica L. , Schnapp Lynn M.
Published: April 2011

The complex interplay between cells and extracellular matrix (ECM) proteins is critical for lung development. Integrins are key modulators of this interaction. The integrin subunit α8 associates with the β1-subunit to form an RGD-binding integrin. We previously showed that, in adult lung, α8 is expressed in contractile interstitial cells and smooth muscle cells and is upregulated in lung injury. To gain insight into the function of α8 during lung development, we examined the spatiotemporal expression of α8 throughout murine lung development. We compared the distribution of α8 with α-smooth muscle actin (αSMA), fibronectin (α8 ligand), and cytokeratin. α8 co-localized with αSMA and fibronectin in the peribronchial and perivascular regions. In all stages, α8 immunoreactivity was detected diffusely in the mesenchyme except for cells surrounding distal, newly forming airways. α8, αSMA, and fibronectin co-localized at tips of secondary septae in the alveolar stage. We conclude that α8 is marker for lung mesenchymal cells starting early in development. α8 is also a marker for smooth muscle cells, expressed as early as αSMA. Co-localization of α8 with fibronectin suggests a role in branching morphogenesis. Furthermore, α8 may participate in secondary septation by modulating signals from the extracellular matrix to alveolar myofibroblasts.
Androgen Regulation of SMR2 Gene Expression in Rat Submandibular Gland: Evidence for a Graded but not a Binary Response
Authors: Huaulmé Jean-François , Courty Yves , Rougeon François , Rosinski-Chupin Isabelle
Published: April 2011

Expression of SMR2, a member of the gene family encoding salivary glutamine/glutamic acid-rich proteins, is regulated by androgens in rat submandibular gland acinar cells. To further characterize SMR2 regulation, we analyzed SMR2 expression during submandibular gland postnatal development and rat puberty at both a global and a single-cell level. Using in situ detection of mature and primary SMR2 transcripts, we show that SMR2 expression is heterogeneous among acinar cells. However, only one cell population with various amounts of mRNAs can be defined. The number of high-expressing cells increases in males during puberty and in females up to 6 weeks of age, suggesting that some factor in addition to acinar differentiation might be important for SMR2 expression in female rats. Involvement of the β-adrenergic system in regulating SMR2 expression was tested in rats exposed daily to isoproterenol for 4 days. Under these conditions we found an increase in SMR2 expression in female rats, associated with an increase in SMR2 mRNA levels in most acinar cells. This suggests that a signaling cascade, elicited by β-adrenergic stimuli, might act in concert with androgens to regulate SMR2 expression.
Three-dimensional Organization of pKi-67: A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold
Authors: Cheutin Thierry , O'Donohue Marie-Françoise , Beorchia Adrien , Klein Christophe , Kaplan Hervé , Ploton Dominique
Published: April 2011

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250–300 nm in diameter, which are themselves composed of 30–50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.
Sex-related Expression of 20α-hydroxysteroid Dehydrogenase mRNA in the Adult Mouse
Authors: Pelletier G. , Luu-The V. , Ren S. Li, L. , Labrie F.
Published: April 2011

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone into its inactive form, 20α-hydroxyprogesterone. To gain information about the exact sites of 20α-HSD mRNA expression, we performed in situ hybridization using a 35S-labeled cRNA probe in tissues of adult mice of both sexes. 20α-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20α-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20α-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20α-HSD mRNA is higher in the female than in the male, suggesting that 20α-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.
C-Met Expression and Mechanical Activation of Satellite Cells on Cultured Muscle Fibers
Authors: Wozniak Ashley C. , Pilipowicz Orest , Yablonka-Reuveni Zipora , Greenway Steven , Craven Shauna , Scott Elliott , Anderson Judy E.
Published: April 2011

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0–6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.
Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development
Authors: Wakisaka Yoshinobu , Furuta Akiko , Masuda Katsuaki , Morikawa Wataru , Kuwano Michihiko , Iwaki Toru
Published: April 2011

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.
Cytokeratin Immunoreactivity in Lobular Intraepithelial Neoplasia
Authors: Bratthauer Gary L. , Miettinen Markku , Tavassoli Fattaneh A.
Published: April 2011

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34βE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34βE12. We conclude that the reactivity seen in the cells of LIN with 34βE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.
Immunocytochemical Detection of GLUT2 at the Rat Intestinal Brush-border Membrane
Authors: Affleck Julie A. , Helliwell Philip A. , Kellett George L.
Published: April 2011

We have proposed a new model of intestinal sugar absorption in which high sugar concentrations promote rapid insertion of the facilitative transporter GLUT2 into the brush-border membrane so that absorptive capacity is precisely regulated to match dietary intake during the assimilation of a meal. However, location of GLUT2 at the brush border by immunocytochemistry has been problematical. We report that control of rapid GLUT2 trafficking and the use of an antibody to a sequence within the large extracellular loop of GLUT2 permits localization of GLUT2 at the brush border. To reveal brush-border GLUT2 fully, it is necessary to digest the sugar chain at the glycosylation site close to the antigenic site. In this way, we have demonstrated by immunocytochemistry PKC-dependent changes in the regulation of brush-border GLUT2 in rat jejunum that correspond to those seen by Western blotting. The functional and immunocytochemical data are now reconciled.
Identification of c-Jun as bcl-2 Transcription Factor in Human Uterine Endometrium
Authors: Li ZL , Abe H. , Ueki K. , Kumagai K. , Araki R. , Otsuki Y.
Published: April 2011

We describe the application of the biomolecular interaction (BIA) technique to detection of the interaction between protein (e.g., c-Jun) and DNA (e.g., two AP-1 motifs from bcl-2 promoter), compared with immunohistochemistry (IHC) of c-Jun. The specific binding assay for the interaction of c-Jun and activating protein-1 (AP-1) motifs was performed using a Biacore 2000 system. Intense immunoreactivity of c-Jun in glandular cells of the human uterine endometrium was observed in the proliferative phase, while c-Jun in stromal cells was expressed throughout the menstrual cycle. In contrast to the IHC of c-Jun, the specific binding of c-Jun to two separate AP-1 motifs in the bcl-2 promoter region was detected only in nuclear extracts of glandular cells, but not in stromal cells, during the proliferative phase. These results indicate that, while transmitting various signals, c-Jun enhances the transcription level of bcl-2, which in turn keeps glandular cells alive and proliferating in normal human endometrium during the proliferative phase. Moreover, the method involving real-time biomolecular interactions such as DNA–protein binding is novel for the study of transcription factors when combined with IHC.
Differential Cellular and Subcellular Localization of Heme-Binding Protein 23/Peroxiredoxin I and Heme Oxygenase-1 in Rat Liver
Authors: Immenschuh Stephan , Baumgart-Vogt Eveline , Tan Melly , Iwahara Shin-Ichiro , Ramadori Giuliano , Dariush Fahimi H.
Published: April 2011

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A–gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.
Localization of mRNAs Encoding Acetylcholinesterase and Butyrylcholinesterase in the Rat Spinal Cord by Nonradioactive In Situ Hybridization
Authors: Mis Katarina , Mars Tomaz , Jevsek Marko , Brank Martina , Zajc–Kreft Katarina , Grubic Zoran
Published: April 2011

In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in α-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. α-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.
Expression of the High-affinity Choline Transporter CHT1 in Rat and Human Arteries
Authors: Lips Katrin S. , Pfeil Uwe , Reiners Katja , Rimasch Christoph , Kuchelmeister Klaus , Braun-Dullaeus Ruediger C. , Haberberger Rainer V. , Schmidt Rupert , Kummer Wolfgang
Published: April 2011

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibro-blasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29–40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.
Fibronectin Accelerates the Growth and Differentiation of Ameloblast Lineage Cells In Vitro
Authors: Tabata Makoto J. , Matsumura Tatsushi , Fujii Takafumi , Abe Makoto , Kurisu Kojiro
Published: April 2011

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial–mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.
High-pressure Freezing of Isolated Gastric Glands Provides New Insight into the Fine Structure and Subcellular Localization of H+/K+-ATPase in Gastric Parietal Cells
Authors: Sawaguchi Akira , McDonald Kent L. , Forte John G.
Published: April 2011

High-pressure freezing (HPF) is currently the most reliable method to obtain an adequately frozen sample for high-resolution morphological evaluation. Here we applied the HPF technique to isolated rabbit gastric glands to reveal structural evidence that may be correlated with functional activity of gastric parietal cells. This approach provided well-preserved fine structure and excellent antigenicity of several parietal cell proteins. Microtubules were abundant in the cytoplasm and frequently appeared to be associating with tubulovesicles. Interestingly, many electron-dense coated vesicles were apparent around the intracellular canaliculi (IC) of resting parietal cells, consistent with active membrane retrieval from the apical membranes. Immunolabeling of H+/K+-ATPase was evident on the endocytic components (e.g., multivesicular bodies) and tubulovesicles. After histamine stimulation, the parietal cells characteristically showed expanded IC membranes with varied features of their apical microvilli. The labeling density of H+/K+-ATPase was fourfold higher on the IC membrane of stimulated parietal cells than on that of resting parietal cells. Immunolabeling of ezrin was clearly identified on the IC and basolateral membranes of parietal cells, corresponding to their F-actin-rich sites. The present findings provide a new insight into the correlation of cell structure and function in gastric parietal cells.
Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium
Authors: Feng Dian , Nagy Janice A. , Pyne Kathryn , Dvorak Harold F. , Dvorak Ann M.
Published: April 2011

The distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium has been disputed. Originally reported to be highly concentrated at interendothelial cell contacts, recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. We re-investigated this question with two different murine anti-CD31 antibodies (MEC 13.3 and M-20), using a pre-embedding immunonanogold electron microscopic procedure that allowed precise label quantitation. MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A164). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes of endothelial cells, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither antibody was reactive in CD31-null mice. We conclude that CD31 is distributed over the entire endothelial cell surface, exclusive of specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.
Tissue Distribution of Placental Leucine Aminopeptidase/Oxytocinase During Mouse Pregnancy
Authors: Kobayashi Honami , Nomura Seiji , Mitsui Takashi , Ito Tomomi , Kuno Naohiko , Ohno Yasumasa , Kadomatsu Kenji , Muramatsu Takashi , Nagasaka Tetsuro , Mizutani Shigehiko
Published: April 2011

Placental leucine aminopeptidase (P-LAP), also called oxytocinase, is an enzyme responsible for hydrolyzing oxytocin. This enzyme is identical to cystine aminopeptidase. We examined the tissue distribution of P-LAP in normal adult mice and also in mothers and fetuses during mouse pregnancy using immunohistochemical (IHC) analysis. P-LAP-immunoreactive protein was expressed in various organs in a cell- and gestational stage-dependent manner. In the kidney, P-LAP was located in distal and collecting tubules but not in proximal tubules. The islet of Langerhans in the maternal pancreas stained positively for P-LAP in the periphery in early gestation. This staining pattern changed so that both the periphery and inner cells were positive during mid-gestation and finally only inner cells were positive in late gestation. Among the hematopoietic cells in the fetal liver, only megakaryocytes showed strong expression of P-LAP. The staining intensity increased with gestation on the apical surface of trophoblasts in the placental labyrinth. These data demonstrate that P-LAP is present in a variety of tissues, and its presence is affected by pregnancy and fetal development. Therefore, P-LAP may play a significant role in various physiological processes in non-pregnant, pregnant, and fetal mice.
Post-embedding Double-labeling of Antigen-retrieved Ultrathin Sections Using a Silver Enhancement-controlled Sequential Immunogold (SECSI) Technique
Authors: Goode Nigel P. , Shires Michael , Crellin Doreen M. , Khan Tahir N. , Mooney Andrew F.
Published: April 2011

In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300-μm-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.
Detection of PrPsc on Lymphoid Tissues from Naturally Affected Scrapie Animals: Comparison of Three Visualization Systems
Authors: Monleón Eva , Monzón Marta , Hortells Paloma , Vargas Antonia , Acín Cristina , Badiola Juan José
Published: April 2011

We assessed three different visualization systems used routinely in research and diagnosis of transmissable spongiform encephalopathies (TSEs) to demonstrate whether the methodology applied to immunohistochemical (IHC) examination may alter the results concerning detection of prion protein (PrPsc) in the lymphoreticular system (LRS): avidin-biotin–peroxidase (Vectastain ABC kit; Vector), Envision (DAKO), and catalyzed signal amplification (CSA; DAKO). The study aimed to determine which of these showed the highest sensitivity, with the hope of providing an accurate tool for pathogenesis and preclinical diagnosis research in TSEs. Histological sections from palatine tonsils, spleen, GALT (ileum and ileocecal valve), and lymph nodes from sheep belonging to a Spanish scrapie-positive flock were processed by IHC using L42 as primary antibody. As substrate chromogen, diaminobenzidine was used, and all slides were subjectively assessed by light microscopy. A further study using an image analyzer software system was carried out to confirm that the conclusion provided by microscopic examination was objective. The CSA system showed the highest sensitivity in all cases, increasing both variables assessed: the number of follicles that were PrPsc-positive was detected as well as the intensity of immunostaining in each of them.
Frequency of M-Cadherin-stained Satellite Cells Declines in Human Muscles During Aging
Authors: Sajko Špela , Kubínová Lucie , Cvetko Erika , Kreft Marko , Wernig Anton , Eržen Ida
Published: April 2011

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30-μm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 ± 2.3 years) and six old (70.8 ± 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group ( p<0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.
Expression of MAL2, an Integral Protein Component of the Machinery for Basolateral-to-Apical Transcytosis, in Human Epithelia
Authors: Marazuela Mónica , Acevedo Agustín , García-López María Angeles , Adrados Magdalena , de Marco María C. , Alonso Miguel A.
Published: April 2011

MAL2, an integral membrane protein of the MAL family, is an essential component of the machinery necessary for the indirect transcytotic route of apical transport in human hepatoma HepG2 cells. To characterize the range of human epithelia that use MAL2-mediated pathways of transport, we carried out an immunohistochemical survey of normal tissues using a monoclonal antibody specific to the MAL2 protein. MAL2 expression was detected in specific types of normal epithelial cells throughout the respiratory system, the gastrointestinal and genitourinary tracts, in exocrine and endocrine glands, and in hepatocytes. Many different types of specialized secretory cells, either organized in discrete clusters (e.g., endocrine cells in the pancreas) or in endocrine glands (e.g., prostate), were also positive for MAL2. In addition to epithelial cells, peripheral neurons, mast cells, and dendritic cells were found to express MAL2. For comparison with normal epithelial tissue, different types of renal carcinoma were also analyzed, revealing alterations in MAL2 expression/distribution dependent on the particular histological type of the tumor. Our results allow the prediction of the existence of MAL2-based trafficking pathways in specific cell types and suggest applications of the anti-MAL2 antibody for the characterization of neoplastic tissue.
Regulation of Leptin mRNA and Protein Expression in Pituitary Somatotropes
Authors: McDuffie Iris A. , Akhter Noor , Childs Gwen V.
Published: April 2011

Leptin, the ob protein, regulates food intake and satiety and can be found in the anterior pituitary. Leptin antigens and mRNA were studied in the anterior pituitary (AP) cells of male and female rats to learn more about its regulation. Leptin antigens were found in over 40% of cells in diestrous or proestrous female rats and in male rats. Lower percentages of AP cells were seen in the estrous population (21 ± 7%). During peak expression of antigens, co-expression of leptin and growth hormone (GH) was found in 27 ± 4% of AP cells. Affinity cytochemistry studies detected 24 ± 3% of AP cells with leptin proteins and growth hormone releasing hormone (GHRH) receptors. These data suggested that somatotropes were a significant source of leptin. To test regulatory factors, estrous and diestrous AP populations were treated with estrogen (100 pM) and/or GHRH (2 nM) to learn if either would increase leptin expression in GH cells. To rule out the possibility that the immunoreactive leptin was bound to receptors in somatotropes, leptin mRNA was also detected by non-radioactive in situ hybridization in this group of cells. In estrous female rats, 39 ± 0.9% of AP cells expressed leptin mRNA, indicating that the potential for leptin production was greater than predicted from the immunolabeling. Estrogen and GHRH together (but not alone) increased percentages of cells with leptin protein (41 ± 9%) or mRNA (57 ± 5%). Estrogen and GHRH also increased the percentages of AP cells that co-express leptin mRNA and GH antigens from 20 ± 2% of AP cells to 37 ± 5%. Although the significance of leptin in GH cells is not understood, it is clearly increased after stimulation with GHRH and estrogen. Because GH cells also have leptin receptors, this AP leptin may be an autocrine or paracrine regulator of pituitary cell function.