bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Antiphotobleaching Reagents for use with FluoroNanogold in Correlative Microscopy
Authors: Takizawa Toshihiro , Robinson John M.
Published: April 2011

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO], p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.
New Frontiers in Gold Labeling: Symposium Overview
Authors: Hainfeld James F. , Robinson John M.
Published: April 2011

The Symposium New Frontiers in Gold Labeling was held at the Fifth Joint Meeting of the Japan Society of Histochemistry and Cytochemistry and the United States Histochemical Society. Speakers described technological developments in this area that improved localization of cellular components. Each presentation is summarized in this overview, and complete articles follow that describe these results in more detail.
New Frontiers in Gold Labeling
Authors: Hainfeld James F. , Powell Richard D.
Published: April 2011

Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab′ antibody fragments, making small and stable probes. Silver enhancement then makes these small gold particles easily visible by EM, LM, and directly by eye. Another advance is the combination of fluorescent and gold probes for correlative microscopy. Chemical crosslinking of gold particles to many biologically active molecules has made possible many novel probes, such as gold-lipids, gold-Ni-NTA, and gold-ATP.
FluoroNanogold Is a Bifunctional Immunoprobe for Correlative Fluorescence and Electron Microscopy
Authors: Takizawa Toshihiro , Robinson John M.
Published: April 2011

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for highresolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.
Ultrastructural Localization of the Vascular Permeability Factor/Vascular Endothelial Growth Factor (VPF/VEGF) Receptor-2 (FLK-1, KDR) in Normal Mouse Kidney and in the Hyperpermeable Vessels Induced by VPF/VEGF-expressing Tumors and Adenoviral Vectors
Authors: Feng Dian , Nagy Janice A. , Brekken Rolf A. , Pettersson Anna , Manseau Eleanor J. , Pyne Kathryn , Mulligan Richard , Thorpe Philip E. , Dvorak Harold F. , Dvorak Ann M.
Published: April 2011

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.
Localization of Enteroviral Antigen in Myocardium and Other Tissues from Patients with Heart Muscle Disease by an Improved Immunohistochemical Technique
Authors: Zhang Hongyi , Li Yanwen , Peng Tianqing , Aasa Malen , Zhang Li , Yang Yingzhen , Archard Leonard C.
Published: April 2011

SUMMARY The association of enterovirus infection and heart muscle diseases has been investigated extensively by detection of viral genomic RNA using nucleic acid hybridization and the reverse transcription-polymerase chain reaction. To further understand the role of enterovirus and its persistence in these diseases, an immunohistochemical technique was optimized to investigate the expression of viral capsid proteins in situ. A monoclonal antibody (5-D8/1) against an epitope in the N-terminus of capsid protein VP1, conserved in the enterovirus genus, was employed. To enhance sensitivity, the EnVison system was used to detect antigen-antibody complex. VP1 was detected in formalin-fixed, paraffin-embedded endomyocardial biopsy or postmortem myocardial tissues and in liver, spleen, lung, kidney, and pancreas from patients with myocarditis or dilated cardiomyopathy, but not from controls. VP1 was localized in cytoplasm of myofibers, often adjacent to necrosis and infiltrate in myocarditis, and was clustered or scattered in dilated cardiomyopathy. This technique can be used for a definitive laboratory diagnosis of enterovirus-associated diseases and for studying the mechanisms of virus persistence in chronic myocardial disease.
Differential Expression of Peroxisome Proliferator-activated Receptors (PPARs) in the Developing Human Fetal Digestive Tract
Authors: Huin Cécile , Corriveau Lina , Bianchi Arnaud , Keller Jean Marie , Collet Philippe , Krémarik-Bouillaud Pascaline , Domenjoud Lionel , Bécuwe Philippe , Schohn Hervé , Ménard Daniel , Dauça Michel
Published: April 2011

SUMMARY We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (α, β, and α) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARα, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARα was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARα, PPARβ, and PPARα exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARα is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.
Fiber Type Composition of the Vastus Lateralis Muscle of Young Men and Women
Authors: Staron Robert S. , Hagerman Fredrick C. , Hikida Robert S. , Murray Thomas F. , Hostler David P. , Crill Mathew T. , Ragg Kerry E. , Toma Kumika
Published: April 2011

SUMMARY This study presents data collected over the past 10 years on the muscle fiber type composition of the vastus lateralis muscle of young men and women. Biopsies were taken from the vastus lateralis muscle of 55 women (21.2 ± 2.2 yr) and 95 men (21.5 ± 2.4 yr) who had volunteered to participate in various research projects. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were classified using mATPase histochemistry, and cross-sectional area was measured for the major fiber types (I, IIA, and IIB). Myosin heavy chain (MHC) content was determined electrophoretically on all of the samples from the men and on 26 samples from the women. With the exception of fiber Type IC, no significant differences were found between men and women for muscle fiber type distribution. The vastus lateralis muscle of both the men and women contained approximately 41% I, 1% IC, 1% IIC, 31% IIA, 6% IIAB, and 20% IIB. However, the cross-sectional area of all three major fiber types was larger for the men compared to the women. In addition, the Type IIA fibers were the largest for the men, whereas the Type I fibers tended to be the largest for the women. Therefore, gender differences were found with regard to the area occupied by each specific fiber type: IIA>I>IIB for the men and I>IIA>IIB for the women. These data establish normative values for the mATPase-based fiber type distribution and sizes in untrained young men and women.
Distribution and Cellular Localization of Prostacyclin Synthase in Human Brain
Authors: Siegle Isabel , Klein Thomas , Zou Ming-Hui , Fritz Peter , Kömhoff Martin
Published: April 2011

SUMMARY Prostacyclin (PGI2) is a labile, lipid-derived metabolite of arachidonic acid synthesized through the sequential action of cyclo-oxygenase (COX) and prostacyclin synthase (PGIS). In addition to its well-characterized vasodilatory and thrombolytic effects, an increasing number of studies report an important role of PGI2 in nociception in various animal species. In this study we investigated the regional distribution of PGIS in human brain by immunohistochemistry and in situ hybridization. PGIS-immunoreactive (ir) protein was localized to blood vessels throughout the brain. Neuronal cells and glial cells, such as microglia and oligodendrocytes, also showed intense labeling. The strongest expression of PGIS was seen in large principal neurons, such as pyramidal cells of the cortex, pyramidal cells of the hippocampus, and Purkinje cells of the cerebellum. Abundance of PGIS mRNA was observed in blood vessels and large neurons and correlated well with the immunohistochemical findings. The expression of PGIS in human brain was further demonstrated by immunoblotting and detection of 6-keto-PGF1α, the stable degradation product of prostacyclin in human brain homogenate. These results demonstrate a widespread expression of PGIS in the central nervous system and suggest a potentially important role of prostacylin in modulating neuronal activity in human brain.
Fluctuating Gene Expression and Localized Cellular Distribution of Vasoactive Intestinal Contractor (VIC) in Mouse Uterus
Authors: Uchide Tsuyoshi , Masuda Hiromi , Lee Yun-Sik , Makiyama Yasushi , Mitsui Youji , Saida Kaname
Published: April 2011

SUMMARY To understand the physiological roles of vasoactive intestinal contractor (VIC) and endothelin-2 (ET-2) in the uterus, we examined the expression levels of VIC mRNA by real-time quantitative reverse transcription-linked polymerase chain reaction (RT-PCR) and characterized the cellular distribution of VIC peptide and mRNA by immunostaining and in situ hybridization in mouse uterus. In pregnant mouse uterus, VIC mRNA expression changed considerably between Days 10.5 and 12.5 of pregnancy. The expression levels were significantly ( p<0.05) higher (approximately fivefold) in the later stage of pregnancy (Days 12.5-17.5) than in the earlier stage (Days 7.5-10.5). In nonpregnant uterus, VIC mRNA expression was significantly ( p<0.05) higher (approximately threefold) in proestrus and estrus than in diestrus. Immunohistochemical studies demonstrated the presence of VIC peptide in endometrial epithelial cells, myometrial cells, and vascular smooth muscle cells during the estrous cycle and pregnancy and after parturition. Notably, myometrial cells showed dominant immunostaining in proestrus and estrus, in the later pregnancy stage, and in the early postpartum period, analogous to the expression pattern of VIC mRNA. In situ hybridization confirmed localization of VIC mRNA in myometrial cells. These findings suggest that VIC may play an important role in the function of myometrial cells.
Developmentally Regulated Alternative Splicing of the α1(XI) Collagen Chain: Spatial and Temporal Segregation of Isoforms in the Cartilage of Fetal Rat Long Bones
Authors: Morris Nicholas P. , Oxford Julia T. , Davies Gillian B.M. , Smoody Barbara F. , Keene Douglas R.
Published: April 2011

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the α1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of α1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.
Distribution of IGFBP-rP1 in Normal Human Tissues
Authors: Degeorges Armelle , Wang Fuan , Frierson Henry F. , Seth Arun , Sikes Robert A.
Published: April 2011

IGFBP-rP1/mac25 is a recently described member of the insulin-like growth factor binding protein (IGFBP) family. It has structural homology to the other members of the IGFBP family but has a lower affinity for insulin-like growth factors (IGFs). In previous studies using RNA blot hybridization, it was shown that the expression of IGFBP-rP1/mac25 was ubiquitous in normal human tissues. In this report we show by immunohistochemistry that the expression of IGFBP-rP1/mac25 is actually restricted to certain organs and specific cell types. We used an antibody raised against a decapeptide of the C-terminal part of the protein that recognizes a ≍37-kD protein under reduced conditions. The immunohistochemistry performed on normal human tissues showed a ubiquitous intense staining of peripheral nerves and a variable degree of positive staining in smooth muscle cells, including those from blood vessel walls, gut, bladder, and prostate. Cilia from the respiratory system, epididymis, and fallopian tube showed intense immunoreactivity. Most endothelial cells showed some positivity, whereas fat cells, plasma cells, and lymphocytes were negative. There was specific expression limited to certain cell types in the kidney, adrenal gland, and skeletal muscle, indicating a possible specialized function of IGFBP-rP1/mac25 in these organs. We further noted an opposite pattern of staining in the lining epithelium of breast (typically positive) and prostate glands (largely negative). The specific localization of IGFBP-rP1/mac25 as described implies a function of the protein. However, its regulation within the IGF axis or a possible direct action of IGFBP-rP1/mac25 remains to be demonstrated.
Confocal Laser Scanning Microscopy of Rat Follicle Development
Authors: Zucker Robert M. , Keshaviah Aparna P. , Price Owen T. , Goldman Jerome M.
Published: April 2011

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-D) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.
Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney
Authors: Weiergräber Marco , Pereverzev Alexey , Vajna Rolf , Henry Margit , Schramm Martin , Nastainczyk Wolfgang , Grabsch Heike , Schneider Toni
Published: April 2011

The calcium channel α1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of α1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac α1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to α1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of α1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of α1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of α1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of α1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin.
Applicability of Different Antibodies for Immunohistochemical Localization of CFTR in Sweat Glands from Healthy Controls and from Patients with Cystic Fibrosis
Authors: Claass Andreas , Sommer Martin , de Jonge Hugo , Kälin Nanette , Tümmler Burkhard
Published: April 2011

The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, ΔF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three—M3A7, MATG 1104, and cc24—met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of ΔF508 CFTR. The antibodies exhibited different sensitivities for detecting ΔF508 CFTR.
Distribution of NADPH-diaphorase and Nitric Oxide Synthase (NOS) in Different Regions of Porcine Oviduct During the Estrous Cycle
Authors: Gawronska Barbara , Bodek Gabriel , Ziecik Adam J.
Published: April 2011

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts ( n=16) on different days of the estrous cycle. One fallopian tube was used for histo-and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units ± SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 ± 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 ± 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 ± 2.5; ampulla 35.6 ± 4.2; infundibulum 24.7 ± 0.8) and then decreased during the remaining stages of the estrous cycle ( p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.
Applications of Plastic Embedding to Electron Microscopic Immunocytochemistry and In Situ Hybridization in Observations of Production and Secretion of Peptide Hormones
Authors: Osamura R. Yoshiyuki , Itoh Yoshiko , Matsuno Akira
Published: April 2011

Plastic embedding has been used to localize various antigens in conjunction with immunohistochemistry. Peptide hormones have been among the antigens that have been studied extensively. Recent application of water-soluble plastics such as LR White and Lowicryl has extended the ranges of detectable antigens and enabled the observation of antigen–antigen or mRNA–antigen combinations. This review article deals with technical aspects, procedures, and applications in endocrine cells.
Placental Expression and Chromosomal Localization of the Human Gcm 1 Gene
Authors: Nait–Oumesmar Brahim , Copperman Alan B. , Lazzarini Robert A.
Published: April 2011

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length h Gcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.
Immunolocalization of Tenascin-C, α9 Integrin Subunit, and αvβ6 Integrin During Wound Healing in Human Oral Mucosa
Authors: Häkkinen Lari , Hildebrand H. Christopher , Berndt Alexander , Kosmehl Hartwig , Larjava Hannu
Published: April 2011

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal–epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-CL) and the putative TN-C-binding α9 and αvβ6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for α9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-CL were localized in the matrix between and underneath α9-expressing epithelial cells. In parallel with gradual downregulation of α9 integrin immunoreactivity in 7-day and older wounds, the expression of αvβ6 integrin was temporarily induced. Integrin αvβ6 co-localized in the same area as TN-C and TN-CL immunoreactivity at the cell–cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-CL were abundantly localized in the granulation tissue. The findings show that TN-CL is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of α9 integrin in migrating epithelial cells and of αvβ6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.
The α4 Isoform of the Na,K-ATPase Is Expressed in the Germ Cells of the Testes
Authors: Blanco Gustavo , Sánchez Gladis , Melton Roger J. , Tourtellotte Warren G. , Mercer Robert W.
Published: April 2011

In addition to the three isoforms of the catalytic subunit of the Na,K-ATPase originally identified (α1, α2, and α3), a fourth α polypeptide (α4) has recently been found in mammalian cells. This novel α-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, α4 is functionally active and comprises approximately half of the Na,K-ATPase activity of the organ. At present, the pattern of expression of the α4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the α4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed α1 polypeptide. The characteristic localization of α4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na,K-ATPase α4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase α4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes. (J Histochem Cytochem 48:1023–1032, 2000)
Glioma-associated Antigen Expression in Oligodendroglial Neoplasms: Tenascin and Epidermal Growth Factor Receptor
Authors: McLendon Roger E. , Wikstrand Carol J. , Matthews Mark R. , Al-Baradei Raid , Bigner Sandra H. , Bigner Darell D.
Published: April 2011

Epidermal growth factor receptor (EGFR), its variant, EGFRvIII, and tenascin are glioma-associated antigens that are hyperexpressed by neoplastic glial cells relative to normal brain, making them attractive antigenic targets for immunotherapy. Preliminary surveys indicate that oligodendroglial tumors also produce these proteins, although the exact patterns and degrees of reactivity are not known. In this study we examined the immunoreactivity of tenascin among 50 oligodendroglial tumors, including 25 well-differentiated oligodendrogliomas (WDOs) and 12 glioblastomas (GBMs) exhibiting high proportions of oligodendroglia-like cells. We used well-characterized immunoreagents with defined specificities against the target antigens on formalin-fixed, paraffin-embedded archival tissue. The tumors were graded according to WHO guidelines. Immunoreactivity was reported on a 1–3 scale according to staining intensity multiplied by a 1–3 distribution scale distribution within tumor as focal (1), multifocal (2), and diffuse (3) for both the parenchymal and the perivascular components. Although there is considerable overlap in antigen production among the grades of tumor, this study establishes the production of tenascin and wild-type EGFR (but not EGFR vIII) in oligodendroglial neoplasms and supports the concept that antigen production increases with tumor grade. (J Histochem Cytochem 48:1103–1110, 2000)
Expression of Neuronal Nitric Oxide Synthase in Several Cell Types of the Rat Gastric Epithelium
Authors: Garcáa–Vitoria Montserrat , Garcáa–Corchón Carmen , Rodráguez José A. , Garcáa–Amigot Fermán , Burrell Maráa A.
Published: April 2011

The aim of this study was to identify which cell types of the rat gastric epithelium express neuronal nitric oxide synthase (nNOS) because the results of the previous studies have been very divergent regarding this point. By the combination of immunohistochemical (IHC) and in situ hybridization (ISH) techniques, we detected expression of nNOS in chief and mucosecretory cells of the gastric epithelium. Moreover, some gastric endocrine cells were immunoreactive for nNOS, although they could not be distinguished in sections treated with ISH techniques. The strongest signal for all antibodies in IHC techniques was obtained when microwave (MW) heating was performed before the IHC procedure. Our results indicate that in the gastric epithelium a variety of cell types are able to produce NO. The NO produced by the different cell types (chief, mucous, and endocrine) may form a complex network of paracrine communication with an important role in gastric physiology. (J Histochem Cytochem 48:1111–1119, 2000)
Still More Complexity in Mammalian Basement Membranes
Authors: Erickson Anna C. , Couchman John R.
Published: April 2011

At the epithelial/mesenchymal interface of most tissues lies the basement membrane (BM). These thin sheets of highly specialized extracellular matrix vary in composition in a tissue-specific manner, and during development and repair. For about two decades it has been apparent that all BMs contain laminins, entactin-1/nidogen-1, Type IV collagen, and proteoglycans. However, within the past few years this complexity has increased as new components are described. The entactin/nidogen (E/N) family has expanded with the recent description of a new isoform, E/N-2/osteonidogen. Agrin and Type XVIII collagen have been reclassified as heparan sulfate proteoglycans (HSPGs), expanding the repertoire of HSPGs in the BM. The laminin family has become more diverse as new α-chains have been characterized, increasing the number of laminin isoforms. Interactions between BM components are now appreciated to be regulated through multiple, mostly domain-specific mechanisms. Understanding the functions of individual BM components and their assembly into macromolecular complexes is a considerable challenge that may increase as further BM and cell surface ligands are discovered for these proteins.
Age-related Alteration of Gap Junction Distribution and Connexin Expression in Rat Aortic Endothelium
Authors: Yeh Hung-I , Chang Hao-Min , Lu Wen-Wei , Lee Yi-Nan , Ko Yu-Shien , Severs Nicholas J. , Tsai Cheng-Ho
Published: April 2011

We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and ≥20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week ( p < 0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until ≥20 months of age (≥20 months vs 28 days, p < 0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages ( p < 0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.
Heterogeneous Distribution of Isoactins in Cultured Vascular Smooth Muscle Cells Does Not Reflect Segregation of Contractile and Cytoskeletal Domains
Authors: Song Jian , Worth Nathalie F. , Rolfe Barbara E. , Campbell Gordon R. , Campbell Julie H.
Published: April 2011

We have previously demonstrated that α-smooth muscle (α-SM) actin is predominantly distributed in the central region and β-non-muscle (β-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, α-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of β-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and α-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of α-SM actin in the central portion of stress fibers was related to continuous myosin and punctate α-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with α-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.
Transduction of TAT-HA-β-galactosidase Fusion Protein into Salivary Gland-derived Cells and Organ Cultures of the Developing Gland, and into Rat Submandibular Gland in Vivo
Authors: Barka Tibor , Gresik Edward W. , Noen Hendrika van der
Published: April 2011

We have studied the transduction of TAT-HA-β-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6–21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate β-galactosidase activity. Transduction of the fusion protein into A5 and C6–21 cells was concentration- and time-dependent. Therefore, the intensity of the β-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-β-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for β-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.
Regional Expression and Histological Localization of Cysteine Sulfinate Decarboxylase mRNA in the Rat Kidney1
Authors: Reymond Isabelle , Bitoun Marc , Levillain Olivier , Tappaz Marcel
Published: April 2011

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of the pathway that forms taurine, a putative osmolyte in the kidney, which was previously localized in various segments of the nephron. Although CSD is known to be expressed in whole kidney extracts, no information on CSD mRNA regional expression and histological localization is yet available. Western blotting and Northern blotting were performed in four dissected regions of the kidney using an antiserum against recombinant CSD and a [32P]-dCTP-labeled CSD cDNA probe, respectively. In situ hybridization was carried out using a [35S]-CTP-labeled CSD RNA probe. A single protein (53 kD) and a single mRNA (2.5 kb) were detected, both of which appeared to be most enriched in the outer stripe of the outer medulla. In situ hybridization of CSD mRNA showed strong labeling of the thick tubules in the outer stripe of the outer medulla and in cortical medullary rays that corresponded to the proximal straight tubules. The significance of this restricted expression of CSD is discussed in relationship to the data previously reported on the location of taurine and the location of the taurine transporter along the nephron.
Immunohistochemical Detection of Ribosomal Transcription Factor UBF and AgNOR Staining Identify Apoptotic Events in Neoplastic Cells of Hodgkin's Disease and in Other Lymphoid Cells
Authors: Torres-Montaner Antonio , Bolívar Jorge , Astola Antonio , Gimenez-Mas Jose A. , Brieva Jose A. , Valdivia Manuel M.
Published: April 2011

Ribosomal RNA synthesis is a key molecular process for understanding the mechanisms that drive cell proliferation. In this process, the upstream binding factor (UBF) is involved in regulating rDNA transcription at the nucleolus, together with RNA polymerase I. Recently, UBF was demonstrated to be a substrate for selective cleavage by specific proteases during apoptosis. Here we studied the expression of UBF in several cases of Hodgkin's disease (HD) by immunostaining and found it to be absent or clearly diminished in a high proportion of Reed–Sternberg cells and Hodgkin cells compared to small reactive lymphocytes. This result contrasted with labeling of those cells by the AgNOR technique, a marker of cell proliferation dependent on increased amounts of several proteins related to ribosome assembly. Disappearance of UBF and preservation of other NOR proteins is consistent with the pattern of selective proteolysis by caspases described in early stages of apoptosis. This correlates well with our results observed on induction of apoptosis in Jurkat cells treated with anti-FAS/APO-1 serum and with those in aged germinal center B-cells, in which UBF was no longer seen although the staining signal of other NOR proteins was maintained. These results support the concept that the rate of apoptosis is higher in neoplastic cells of HD than in the benign reactive lymphocyte population. Differential proteolysis of NOR proteins, as revealed by double staining of UBF and AgNOR, may prove valuable for identification of early stages of apoptosis in cytological and histopathological samples.
Novel Method to Quantify Neuropil Threads in Brains from Elders With or Without Cognitive Impairment
Authors: Mitchell Thomas W. , Nissanov Jonathan , Han Li-Ying , Mufson Elliott J. , Schneider Julie A. , Cochran Elizabeth J. , Bennett David A. , Lee Virginia M.-Y. , Trojanowski John Q. , Arnold Steven E.
Published: April 2011

Pathological alterations in dendrites and axons (i.e., neuritic pathologies) occur in the normal aging brain as well as in brains from elders with mild cognitive impairment and neurodegenerative dementia. These alterations may correlate with clinical measures of cognitive abilities, but the contribution of neuropil threads (NTs), which constitute 85–90% of cortical tau pathology, has not been clear because of the lack of quantitative methodologies. We combined quantitative fractionation and image analysis to devise a strategy for measuring the burden of tau-rich NTs in the entorhinal and perirhinal cortex of brains from elders with and without cognitive impairment, including dementia due to Alzheimer's disease (AD). On the basis of data presented here using this novel strategy, we conclude that this quantitative imaging technique will facilitate efforts to determine the behavioral correlations of neuritic lesions in AD and other brain disorders.
Gender Differences and the Effect of Different Endocrine Situations on the NOS Expression Pattern in the Anterior Pituitary Gland
Authors: González-Hernández Tomás , González M. Carmen
Published: April 2011

The presence of neuronal nitric oxide synthase (nNOS) in two populations of pituitary cells, gonadotrophs (LH) and folliculostellate (FS) cells, suggests that pituitary nitric oxide (NO) is involved in the control of hormone secretion. We have used single and double immunostaining and quantitative procedures to investigate possible gender-related differences in the nNOS expression pattern in the anterior pituitary lobe and its possible alterations in different endocrine situations. Our results reveal a sexual dimorphism in the pattern of nNOS expression. In males, nNOS is mainly found in FS cells, whereas only a few LH cells express nNOS. Conversely, in females, nNOS is mainly found in LH cells. After gonadectomy, paralleling an increase in LH cell size and serum luteinizing hormone (LH) levels, there is nNOS upregulation in LH cells and nNOS downregulation in FS cells. After testoterone replacement, LH cells become nNOS-immunonegative again. In lactating rats, LH cells overexpress nNOS, but LH cell size and serum LH levels are low. This suggests that, depending on its cellular source, pituitary NO can exert either an inhibitory or a stimulatory effect on hormone secretion. When released from FS cells, NO exerts a paracrine inhibitory effect, and when released from gonadotrophs it exerts an autocrine or paracrine stimulatory effect on LH or prolactin secretion, respectively.
Developmental Mucin Gene Expression in the Gastroduodenal Tract and Accessory Digestive Glands. II. Duodenum and Liver, Gallbladder, and Pancreas
Authors: Buisine Marie-Pierre , Devisme Louise , Degand Pierre , Dieu Marie-Claire , Gosselin Bernard , Copin Marie-Christine , Aubert Jean-Pierre , Porchet Nicole
Published: April 2011

Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6.5–27 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatio–temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.