bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Galectin-1 and Galectin-3 in Human Fetal Thyroid Gland
Authors: Savin Svetlana B. , Cvejić Dubravka S. , Janković Miroslava M.
Published: April 2011

High levels of expression of galectin-1 and galectin-3, the β-galactoside-binding proteins, have been recently described in malignant thyroid tumors but not in adenomas nor in normal thyroid tissue. However, there are no data about the expression of these galectins during fetal thyroid development. In this study we analyzed immunohistochemically the presence of galectin-1 and galectin-3 in human fetal thyroid glands (16–37 weeks of gestation). Weak to moderate cytoplasmic staining for galectin-1 was observed in follicular cells of all fetal thyroids. Galectin-3 could not be detected in thyroid follicular cells of any fetal thyroid investigated. Both galectins were detected in stromal tissue, but staining for galectin-1 was more intense. The absence of galectin-3 in thyroid cells during fetal development suggests that galectin-3 is expressed de novo during malignant transformation of thyroid epithelium, and that galectin-1 could be considered an oncofetal antigen. The results obtained indicated potential roles for galectin-1 and galectin-3 during the investigated period of human fetal thyroid gland development. Both galectins might participate in developmental processes regarding stromal fetal thyroid tissue organization, whereas galectin-1 might have a function in thyroid epithelium maturation.
Expression of Hornerin in Stratified Squamous Epithelium in the Mouse: A Comparative Analysis with Profilaggrin
Authors: Makino Teruhiko , Takaishi Mikiro , Toyoda Masahiko , Morohashi Masaaki , Huh Nam-ho
Published: April 2011

We have recently identified a novel protein named hornerin, the structural features of which are most similar to those of profilaggrin, an essential protein for keratinization of epidermal tissues. In this study we examined the expression of hornerin compared with that of profilaggrin in various mouse tissues. Hornerin was expressed in the upper epidermis of newborn mouse skin, as was profilaggrin. In addition, both hornerin and profilaggrin were expressed in the tongue, esophagus, and forestomach. In all four tissues, immunostaining for hornerin and profilaggrin showed a granular pattern, and most of the signals for the two proteins were co-localized on keratohyalin granules. This was confirmed by double immunoelectron microscopy. Within keratohyalin granules, hornerin was detected more frequently in the periphery, whereas profilaggrin was equally distributed. A quantitative RT-PCR revealed that both genes were expressed at highest levels in the forestomach and at the next highest levels in skin. Profilaggrin mRNA was most abundant in the forestomach. In skin, the amount of hornerin mRNA was more than fourfold greater than the amount of profilaggrin mRNA. These results form the basis for a better understanding of possible overlapping and/or differential functions of hornerin and profilaggrin.
Human Kallikrein 13 Expression in Normal Tissues: An Immunohistochemical Study
Authors: Petraki Constantina D. , Karavana Vassiliki N. , Diamandis Eleftherios P.
Published: April 2011

The human tissue kallikrein 13 gene (KLK13), encoding for hK13 protein, was recently cloned and characterized. Here we describe the immunohistochemical (IHC) localization of hK13 in normal human tissues and compare it with the expression of two other kallikreins, hK6 and hK10. We performed the streptavidin-biotin IHC method on 204 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue, using a polyclonal and a monoclonal hK13 antibody. The staining was cytoplasmic and both antibodies yielded similar results. The hK13 protein was revealed in a variety of tissues, mainly in glandular epithelia. Other epithelia that expressed hK13 included the urothelium, the spermatic epithelium, and the epithelium of the choroid plexus. hK13 was intensely immunoexpressed by some endocrine organs, such as the adenohypophysis, the thyroid gland, the parathyroid glands, the adrenal medulla, the Leydig cells of the testis, and the cells of the endocrine pancreas. Immunoreactivity was also observed in the primordial follicles, the corpus luteum, and sparse luteinized cells in the stroma of the ovary, the trophoblastic cells of the placenta, the Hassall's corpuscles of the thymus, and chondrocytes. Nerves and ganglia of the peripheral nervous system, and both neurons and glial cells in the central nervous system, were positive. In short, hK13 was expressed by many glandular epithelia, some endocrine organs, and some specialized epithelia and cells. Comparison of these data with hK6 and hK10 expression suggests that the three kallikreins have a similar IHC pattern in normal human tissues.
New Techniques for Whole-mount NADPH-diaphorase Histochemistry Demonstrated in Insect Ganglia
Authors: Ott Swidbert R. , Elphick Maurice R.
Published: April 2011

Fixation-resistant NADPH-diaphorase (NADPHd) activity is used widely as a marker for nitric oxide synthase (NOS). In frozen sections, NADPHd histochemistry yields high anatomic definition. In whole-mounts, however, poor penetration of the reagents, background staining, and tissue opacity severely limit its application. Here we report a combination of new methods that significantly improves whole-mount NADPHd staining. We demonstrate these methods in the thoracic ganglia of a large insect, the locust Schistocerca gregaria, in which NADPHd has been analyzed previously using both whole-mounts and serial section reconstructions. The penetration of the staining reagents was markedly improved after fixation in methanol/formalin compared to phosphate-buffered formaldehyde. Methanol/formalin also reduced nonspecific NADPHd and enhanced the selective staining. Penetration was further enhanced by incubation regimens that exploit the temperature- or pH-dependence of NADPHd. In combination with methanol/formalin fixation, this permitted staining to develop evenly throughout these comparatively large invertebrate ganglia. These improvements were complemented by a new clearing technique that preserves the NADPHd staining, gives excellent transparency, and avoids distortion of specimen morphology. The new methods revealed the three-dimensional architecture of NADPHd expression in locust ganglia in unprecedented detail and may similarly improve wholemount detection of NADPHd in other invertebrate and vertebrate preparations.
The Nuclear Localization of NFκB and p53 Is Positively Correlated with HPV16 E7 Level in Laryngeal Squamous Cell Carcinoma
Authors: Du Jing , Chen George G. , Vlantis Alexander C. , Xu Hu , Tsang Raymond K.Y. , van Hasselt Andrew C.
Published: April 2011

The interaction between the HPV (human papilloma virus) 16 E7 and other cell growth factors, such as p53 and NFκB in laryngeal cancer is not clearly understood. The aim of this study was to examine the expression of these three proteins in tumor and non-tumor laryngeal tissues from patients with laryngeal squamous cell carcinoma. These three proteins were dominantly expressed in the nucleus and their levels were higher in the tumor tissue than in the non-tumor tissue, although the comparison between the tumor and non-tumor tissues of p53 staining did not reach significance. The intensity of the nuclear stain of E7 and p53 was stronger than that of p65, a subunit of NFκB. Correlation analysis revealed that there was a positive relationship between the level of HPV16 E7 and the expression of p65. The correlation between E7 and p53 was also significant, although to a lesser degree. The finding of nuclear localization of p65 suggests that NFκB is constantly activated in the laryngeal cancer cells, whereas the sequestration of p53 in the nucleus may represent a mutated form of p53, which is probably inactivated by HPV16 oncoproteins. In conclusion, this study suggests that the nuclear localization of NFκB and p53 may play a role in the development of human laryngeal squamous cell carcinoma infected with HPV16.
Localization of the BiP Molecular Chaperone with Respect to Endoplasmic Reticulum Foci Containing the Cystic Fibrosis Transmembrane Conductance Regulator in Yeast
Authors: Sullivan Mara L. , Youker Robert T. , Watkins Simon C. , Brodsky Jeffrey L.
Published: April 2011

Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.
Proteotyping of Mammary Tissue from Transgenic and Gene Knockout Mice with Immunohistochemical Markers: a Tool To Define Developmental Lesions
Authors: Shillingford Jonathan M. , Miyoshi Keiko , Robinson Gertraud W. , Bierie Brian , Cao Yixue , Karin Michael , Hennighausen Lothar
Published: April 2011

Through the use of transgenic and gene knockout mice, several studies have identified specific genes required for the functional development of mammary epithelium. Although histological and milk protein gene analyses can provide useful information regarding functional differentiation, they are limited in their ability to precisely define the molecular lesions. For example, mice that carry a mutation in one of the subunits of the IκB kinase, IKKα, cannot lactate despite the presence of histologically normal alveolar compartment and the expression of milk protein genes. To further define and understand such lesions on a molecular level, we sought evidence for proteins that are differentially expressed during mammary gland development with a view to generating a tissue proteotype. Using database screens and immunohistochemical analyses, we have identified three proteins that exhibit distinct profiles. Here, using mouse models as test biological systems, we demonstrate the development and application of mammary tissue proteotyping and its use in the elucidation of specific developmental lesions. We propose that the technique of proteotyping will have wide applications in the analyses of defects in other mouse models.
Same Serial Section Correlative Light and Energy-filtered Transmission Electron Microscopy
Authors: Ren Ying , Kruhlak Michael J. , Bazett-Jones David P.
Published: April 2011

Correlative imaging of a specific cell with both the light microscope and the electron microscope has proved to be a difficult task, requiring enormous amounts of patience and technical skill. We describe a technique with a high rate of success, which can be used to identify a particular cell in the light microscope and then to embed and thin-section it for electron microscopy. The technique also includes a method to obtain many uninterrupted, thin serial sections for imaging by conventional or energy-filtered transmission electron microscopy, to obtain images for 3D analysis of detail at the suborganelle level.
Co-localization of the Mammalian Hemochromatosis Gene Product (HFE) and a Newly Identified Transferrin Receptor (TfR2) in Intestinal Tissue and Cells
Authors: Griffiths William J.H. , Cox Timothy M.
Published: April 2011

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown. Using specific peptide antisera, we examined the duodenal localization of HFE and TfR2 in humans and mice, with and without HFE deficiency, by confocal microscopy. We also investigated potential interactions of these proteins in human intestinal cells in situ. Duodenal expression of HFE and TfR2 (but not TfR1) in wild-type mice and humans was restricted to crypt cells, in which they co-localized. HFE deficiency disrupted this interaction, altering the cellular distribution of TfR2 in human crypts. In human Caco-2 cells, HFE and TfR2 co-localized to a distinct CD63-negative vesicular compartment showing marked signal enhancement on exposure to iron-saturated transferrin ligand, indicating that HFE preferentially interacts with TfR2 in a specialized early endosomal transport pathway for transferrin-iron. This interaction occurs specifically in small intestinal crypt cells that differentiate to become iron-absorbing enterocytes. Our immunohistochemical findings provide evidence for a novel mechanism for the regulation of iron balance in mammals.
An Antibody Raised Against In Vitro-derived Human Mast Cells Identifies Mature Mast Cells and a Population of Cells that are Fc∊RI+, Tryptase−, and Chymase− in a Variety of Human Tissues
Authors: Qi Jian Cheng , Li Lixin , Li Yong , Moore Kate , Madigan Michele C. , Katsoulotos Gregory , Krilis Steven A.
Published: April 2011

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immuno-labeling with 2C7 and tryptase, chymase, Fc∊RIα, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase+ or chymase+ mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc∊RIα. However, there were a significant number of Fc∊RIα+ cells in all tissues studied that were tryptase− and/or chymase−. In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93–96% of the Fc∊RIα+ cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase+ or chymase+ cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase+ cells in colon and stomach were c-kit+. These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc∊RIα+, chymase−, and tryptase− cells in a variety of human tissues.
Expression of Catalase mRNA and Protein in Adult Rat Brain: Detection by Nonradioactive In Situ Hybridization with Signal Amplification by Catalyzed Reporter Deposition (ISH–CARD) and Immunohistochemistry (IHC)/Immunofluorescence (IF)
Authors: Schad Arno , Fahimi H. Dariush , Völkl Alfred , Baumgart Eveline
Published: April 2011

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.
Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I Expression in Gastric Carcinoma Cell Lines
Authors: Marcos Nuno T. , Cruz Andrea , Silva Filipe , Almeida Raquel , David Leonor , Mandel Ulla , Clausen Henrik , Mensdorff-Pouilly Silvia von , Reis Celso A.
Published: April 2011

Mucin O-glycosylation in cancer is characterized by aberrant expression of immature carbohydrate structures leading to exposure of simple mucin-type carbohydrate antigens and peptide epitopes. Glycosyltransferases controlling the initial steps of mucin O-glycosylation are responsible for the altered glycosylation observed in cancer. We studied the expression in gastric cell lines of six UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T1, T2, T3, T4, T6, T11) that catalyze the initial key step in the regulation of mucin O-glycosylation, the transfer of GalNAc from UDP-GalNAc to serine and threonine residues. We also studied the expression of ST6GalNAc-I, the enzyme responsible for the synthesis of Sialyl-Tn antigen (NeuAcα2,6GalNAc) and the ST3Gal-I, the enzyme responsible for the synthesis of Sialyl-T antigen (NeuAcα2,3Galβ1,3GalNAc). This study was done using specific monoclonal antibodies, enzymatic assays, and RT-PCR. Our results showed that GalNAc-T1, -T2, and -T3 have an ubiquitous expression in all gastric cell lines, whereas GalNAc-T4, -T6, and -T11 show a restricted expression pattern. The immunoreactivity with MAb VU-2-G7 suggests that, apart from GalNAc-T4, another GalNAc transferase is involved in the glycosylation of the Thr in the PDTR region of the MUC1 tandem repeat. The expression of ST3Gal-I correlates with the expression of the Sialyl-T antigen in gastric cell lines and in the control cell lines studied. The expression of ST6GalNAc-I is low in gastric cell lines, in accordance with the low/absent expression of the Sialyl-Tn antigen.
In Situ Localization of Gelatinolytic Activity in the Extracellular Matrix of Metastases of Colon Cancer in Rat Liver Using Quenched Fluorogenic DQ-gelatin
Authors: Mook Olaf R.F. , Overbeek Claudia Van , Ackema Eleonora G. , Maldegem Febe Van , Frederiks Wilma M.
Published: April 2011

Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin zymography that the tumors contained MMP-2 and MMP-9, but only MMP-2 was present in the active form. Immunolocalization of MMP-2 showed that the protein was localized at basement membranes of colon cancer cells and in intratumor stroma, associated with extracellular matrix (ECM) components. However, zymography and immunohistochemistry (IHC) do not provide information on the localization of MMP activity. Therefore, we developed an in situ zymography technique using the quenched fluorogenic substrate DQ-gelatin in unfixed cryostat sections. The application of DQ-gelatin in combination with a gelled medium allows precise localization of gelatinolytic activity. Fluorescence due to gelatinolytic activity was found in the ECM of tumors and was localized similarly to both MMP-2 protein and collagen type IV, its natural substrate. The localization of MMP-2 activity and collagen type IV at similar sites suggests a role of MMP-2 in remodeling of ECM of stroma in colon cancer metastases in rat liver.
Stigmoid Bodies Contain Type I Receptor Proteins SorLA/LR11 and Sortilin: New Perspectives on Their Function
Authors: Gutekunst Claire-Anne , Torre Enrique R. , Sheng Zejuan , Yi Hong , Coleman Sarah H. , Riedel I. Björn , Bujo Hideaki
Published: April 2011

Stigmoid bodies (SBs) are structures in the cytoplasm of neurons. SBs are mostly found in the hypothalamic region of the rat and contain a protein called huntingtin-associated protein 1 (HAP1). In a recent publication, large cytoplasmic structures were shown to be immunoreactive for a type I receptor called SorLA/LR11. By light microscopic analysis, these structures appeared similar to SBs in size and in brain regional and subcellular localization. To determine whether these large puncta correspond to HAP1-containing SBs, we used antibodies specific to various domains of the apolipoprotein receptor LR11 to perform immunocytochemistry in rat and mouse brain tissue. Transfection studies using HeLa cells were conducted to demonstrate the specificity of the antibodies. We found that, in both species, antibodies to the domain II (or VSP10 for vacuolar sorting protein 10 domain) of LR11 immunoreact with large cytoplasmic structures. Co-localization immunolabeling experiments in rat brain tissue sections and in neuron cultures showed that these LR11-immunoreactive structures correspond to HAP1-positive SBs. Electron microscopy was performed in rat hypothalamus and further demonstrated the presence of LR11 in SBs and its co-localization with HAP1. LR11-containing SBs were most abundant in the hypothalamus but were also found in many brainstem nuclei, thalamus, and hippocampus. Our data also show that sortilin, another transmembrane protein containing a VPS10 domain, localizes to large cytoplasmic puncta and is found in LR11-positive and Hap1-positive SBs in hypothalamic neuron cultures.
Expression of Gap Junction Protein Connexin43 in the Adult Rat Cochlea: Comparison with Connexin26
Authors: Suzuki Toshihiro , Takamatsu Tetsuro , Oyamada Masahito
Published: April 2011

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.
Detection of Oligodendrocytes in Tissue Sections Using PCR Synthesis of Digoxigenin-labeled Probes
Authors: Jalabi Walid , Cerghet Mirela , Skoff Robert P. , Ghandour M. Said
Published: April 2011

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.
Alterations of Collagen XVII Expression During Transformation of Oral Epithelium to Dysplasia and Carcinoma
Authors: Parikka Mataleena , Kainulainen Tiina , Tasanen Kaisa , Väänänen Anu , Bruckner-Tuderman Leena , Salo Tuula
Published: April 2011

Collagen XVII (BP180) is a hemidesmosomal transmembrane component that has been hypothesized to participate in keratinocyte adhesion and motility. Using immunohistochemical (IHC) and in situ hybridization (ISH) methods, we showed downregulation of collagen XVII in basal cells in mild dysplasias and upregulation in suprabasal keratinocytes in moderate and severe dysplasias as well as in the central cells of grade II and III squamous cell carcinomas (SCCs). Overexpression of collagen XVII was found at the invasive front of the tumors. Collagen XVII and its cleaved ectodomain were characterized from culture extracts and precipitates of oral keratinocytes, tongue carcinoma cells, and tumor tissue extract. Malignant cell lines exhibited increased collagen XVII expression in immunoblotting analysis. In oral keratinocytes, collagen XVII gene expression was significantly induced by PMA but not by the inflammatory cytokines TGF-β1, TNF-α, EGF, IL-1β, and IL-6. These results indicate altered expression of collagen XVII at different stages of carcinogenesis and suggest a correlation between overexpression of collagen XVII and tumor progression. The reduced collagen XVII expression at the early step of carcinogenesis may reflect disturbed keratinocyte adhesion to the basement membrane.
Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model1
Authors: Abboud Sherry L. , Bunegin Maria , Ghosh-Choudhury Nandini , Woodruff Kathleen
Published: April 2011

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a −774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the −774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the −774/+183-bp region driving the E. coli β-galactosidase (lacZ) reporter gene were generated. β-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of β-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the −774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.
Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer
Authors: Murata Shin-ichi , Herman Petr , Mochizuki Kunio , Nakazawa Tadao , Kondo Tetsuo , Nakamura Nobuki , Lakowicz Joseph R. , Katoh Ryohei
Published: April 2011

We employed microscopic intensity-based fluorescence resonance energy transfer (FRET) images with correction by donor and acceptor concentrations to obtain unbiased maps of spatial distribution of the AT- and GC-rich DNA regions in nuclei. FRET images of 137 bovine aortic endothelial cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and corrected for the donor and acceptor concentrations by the Gordon's method based on the three fluorescence filter sets. The corrected FRET images were quantitatively analyzed by texture analysis to correlate the spatial distribution of the AT- and GC-rich DNA regions with different phases of the cell cycle. Both visual observation and quantitative texture analysis revealed an increased number and size of the low FRET efficiency centers for cells in the G2/M-phases, compared to the G1-phase cells. We have detected cell cycle-dependent changes of the spatial organization and separation of the AT- and GC-rich DNA regions. Using the corrected FRET (cFRET) technique, we were able to detect early DNA separation stages in late interphase nuclei.
Fluorogenic Substrate [Ala-Pro]2-Cresyl Violet But Not Ala-Pro-Rhodamine 110 Is Cleaved Specifically by DPPIV Activity: A Study in Living Jurkat Cells and CD26/DPPIV-transfected Jurkat Cells
Authors: Boonacker Emil , Elferink Sjoerd , Bardai Abdennasser , Fleischer Bernard , Noorden Cornelis J.F. Van
Published: April 2011

Fluorogenic substrates [Ala-Pro]2-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro]2-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro]2-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro]2-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro]2-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction.
Expression of Notch1 and Jagged2 in the Enteric Nervous System
Authors: Sander Guy R. , Brookes Simon J.H. , Powell Barry C.
Published: April 2011

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.
HOPE Fixation of Cytospin Preparations of Human Cells for In Situ Hybridization and Immunocytochemistry
Authors: Umland Oliver , Ulmer Artur J. , Vollmer Ekkehard , Goldmann Torsten
Published: April 2011

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES–glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.
Antigen Retrieval Trial for Post-embedding Immunoelectron Microscopy by Heating with Several Unmasking Solutions
Authors: Saito Nagahito , Konishi Kohei , Takeda Hiroshi , Kato Mototsugu , Sugiyama Toshiro , Asaka Masahiro
Published: April 2011

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti- H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in postembedding immunogold electron microscopy.
Characterization of Matriptase Expression in Normal Human Tissues
Authors: Oberst Michael D. , Singh Baljit , Ozdemirli Metin , Dickson Robert B. , Johnson Michael D. , Lin Chen-Yong
Published: April 2011

Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.
All Three Promoters of the Acetyl-Coenzyme A-Carboxylase α-encoding Gene Are Expressed in Mammary Epithelial Cells of Ruminants
Authors: Molenaar Adrian , Mao Jianqiang , Oden Kim , Seyfert Hans-Martin
Published: April 2011

The activity of the enzyme acetyl-CoA-carboxylase α (ACC-α) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-α-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-α synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.
P63 Immunoreactivity Distinguishes Upper Urinary Tract Transitional-cell Carcinoma and Renal-cell Carcinoma Even in Poorly Differentiated Tumors
Authors: Langner Cord , Ratschek Manfred , Tsybrovskyy Oleksiy , Schips Luigi , Zigeuner Richard
Published: April 2011

P63 is essential for the differentiation of normal urothelium and is also expressed in transitional cell carcinoma (TCC) of the bladder. We investigated p63 immunoreactivity in upper urinary tract TCC ( n = 53) and in renal-cell carcinoma (RCC; n = 188) using a tissue microarray technique. P63 expression was detected in 51/53 (96.2%) TCCs, showing decreased expression in high-stage (pT1 and pT2 100%; pT3 90.9%) and poorly differentiated (G1 and G2 100%; G3 92%) tumors. All RCCs were negative for p63. P63 proved to be a helpful tool, even in poorly differentiated and undifferentiated renal malignancies, to distinguish TCC from RCC.
A New Approach to an Unbiased Estimate of the Hepatic Stellate Cell Index in the Rat Liver: An Example in Healthy Conditions1
Authors: Marcos Ricardo , Rocha Eduardo , Henrique Rui M.F. , Monteiro Rogério A.F.
Published: April 2011

Evaluation of activation and proliferation of hepatic stellate cells (HSCs) must be grounded on solid quantitative data under normal conditions. The HSC index (HSCI), number of HSCs per 1000 hepatocytes (HEP), is often used in hepatology but has been never determined using stereology. Systematically sampled sections were immunostained against glial fibrillary acidic protein and carcinoembryonic antigen, allowing unequivocal distinction of HSC and mononuclear/binuclear HEP. With the optical disector the HSCI was estimated as 109 (coefficient of error = 0.04). This work provides a sound technical basis for experiments in which the estimation of HSCI and/or simultaneous quantification of HSC and HEP are relevant.
Impaired Angiogenesis in Aging Is Associated with Alterations in Vessel Density, Matrix Composition, Inflammatory Response, and Growth Factor Expression
Authors: Sadoun Eman , Reed May J.
Published: April 2011

It is generally accepted that angiogenesis is delayed in aging. To define the effects of age on the neovascular response, polyvinyl alcohol sponges were implanted SC in young (6–8 months old, n = 11) and aged (23-25 months old, n = 13) mice and sampled at 14 and 19 days. Angiogenic invasion was significantly delayed in aged mice at 14d relative to young at 14d (% area of invasion 9.0 ± 3.7 vs 19.0 ± 5.6; p = 0.02). Although microvessel morphology and basement membrane composition were similar between the age groups, a significant decrease in capillary density was noted in aged tissues at 14d (7.5 ± 4.1) and 19d (12.1 ± 2.8) relative to young at 14d (18.7 ± 2.3) ( p<0.01 A14d vs Y14d). In comparison to young at 14d, the inflammatory response was decreased by 43 ± 2.9% and 36 ± 7.8% in aged mice at 14d and 19d, respectively. Tissues of aged mice showed less newly deposited collagen. There was a lack of expression of transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in aged mice at 14d (0.63 ± 0.3) and 19d (1.14 ± 0.5) vs young at 14d (1.92 ± 0.5) (p≤0.01 A14d vs Y14d for VEGF). However, similar production of VEGF receptor2 was observed. In contrast to young mice, there was significantly increased expression of thrombospondin-2 (TSP-2) in aged mice from 14d (14.6 × 103 ± 7.3 × 103) to 19d (34.9 × 103 ± 17 × 103). We conclude that angiogenesis in aging is not merely delayed, but is altered due to multiple impairments.
Stimulation of Fos- and Jun-related Genes During Distraction Osteogenesis
Authors: Lewinson Dina , Rachmiel Adi , Rihani-Bisharat Souhir , Kraiem Zaki , Schenzer Pesia , Korem Sigal , Rabinovich Yaron
Published: April 2011

Bone cells respond to mechanical stimulation by gene expression. The molecular events involved in the translation of mechanical stimulation into cell proliferation and bone formation are not yet well understood. We looked for the expression of early-response genes of the AP-1 transcription factor complex in an in vivo bone regeneration system subjected to mechanical forces because these genes were found to be related to mechanotransduction and important for bone development. Sheep maxillary bone was distracted daily for 15 days. c-Jun and c-Fos were evaluated by Northern blotting analysis and immunohistochemistry in biopsy specimens removed at 8 and 15 days and were compared with post-osteotomy but not distracted repair tissue. Elevated levels of c-Jun and c-Fos mRNA were found after 8 days of distraction. Likewise, mesenchyme-like and fibroblast-like cells composing the 8-day distracted regeneration tissue showed increases in the intensity of immunostaining compared to cells in the corresponding non-distracted fracture repair tissue. After 15 days of distraction, when bone trabeculae start to form distally and proximally in the distracted regeneration tissue, mostly preosteoblasts and osteoblasts retained c-Fos and c-Jun immunoreactivity, similar to bone-associated cells in control non-distracted fracture repair tissue. We propose that the elevated expression of c-Jun and c-Fos is related to mechanical stimulation in this in vivo bone regeneration system.
Localization of Carbonic Anhydrase XII to the Basolateral Membrane of H+-secreting Cells of Mouse and Rat Kidney
Authors: Kyllönen Matti S. , Parkkila Seppo , Rajaniemi Hannu , Waheed Abdul , Grubb Jeffrey H. , Shah Gul N. , Sly William S. , Kaunisto Kari
Published: April 2011

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO3− absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40–45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H+-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO3− absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.
Development of Thymic Microenvironments In Vitro Is Oxygen-dependent and Requires Permanent Presence of T-cell Progenitors
Authors: Germeraad Wilfred T.V. , Kawamoto Hiroshi , Itoi Manami , Jiang Yufei , Amagai Takashi , Katsura Yoshimoto , van Ewijk Willem
Published: April 2011

Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.
Intravenous Liposomal Prednisolone Downregulates In Situ TNF-α Production by T-cells in Experimental Autoimmune Encephalomyelitis
Authors: Schmidt Jens , Metselaar Josbert M. , Gold Ralf
Published: April 2011

Multiple sclerosis (MS) relapses are treated with high-dose IV glucocorticosteroids. Here we investigated mechanisms of long-circulating polyethylene glycol-coated liposomes encapsulating prednisolone (PL) in adoptive transfer experimental autoimmune encephalomyelitis. Rats received IV 10 mg/kg PL 6, 18, or 42 hr before sacrifice at disease maximum. In formalin-fixed, paraffin-embedded spinal cord we employed a nonfluorescent immunohistochemical (IHC) double labeling. We stained for tumor necrosis factor-α (TNF-α) in combination with a T-cell antigen. Compared with PBS-containing liposomes, PL at 18 hr, and more at 42 hr, significantly reduced the rate of TNF-α double-labeled T-cells. This correlated with an ameliorated disease score at day 5 after PL 42 hr. Our results help to further understand mechanisms of action of drug targeting by liposomal steroids, with possible implications for treatment of autoimmune disorders such as MS.