Altered Expression of CXCL13 and CXCR5 in Intractable Temporal Lobe Epilepsy Patients and Pilocarpine-Induced Epileptic Rats.

PubMed

Li, Ruohan; Ma, Limin; Huang, Hao; Ou, Shu; Yuan, Jinxian; Xu, Tao; Yu, Xinyuan; Liu, Xi; Yang, Juan; Chen, Yangmei; Peng, Xi

2017-02-01

The mechanisms that underlie the pathogenesis of epilepsy are still unclear. Recent studies have indicated that inflammatory processes occurring in the brain are involved in a common and crucial mechanism in epileptogenesis. C-X-C motif chemokine ligand 13 (CXCL13) and its only receptor, C-X-C motif chemokine receptor 5 (CXCR5), are highly expressed in the central nervous system (CNS) and participate in inflammatory responses. The present study aimed to assess the expression of CXCL13 and CXCR5 in the brain tissues of both patients with intractable epilepsy (IE) and a rat model (lithium-pilocarpine) of temporal lobe epilepsy (TLE) to identify possible roles of the CXCL13-CXCR5 signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical, double-labeled immunofluorescence and Western blot analyses were performed in this study. CXCL13 and CXCR5 mRNA expression and protein levels were found to be significantly up-regulated in the TLE patients and TLE rats. Further, CXCL13 and CXCR5 protein levels were altered during the different epileptic phases after onset of status epilepticus (SE) in the pilocarpine model rats, including the acute phase (6, 24, and 72 h), latent phase (7 and 14 days) and chronic phase (30 and 60 days groups). Moreover, double-labeled immunofluorescence analysis revealed that CXCL13 was mainly expressed in the cytomembranes and cytoplasm of neurons and astrocytes, while CXCR5 was mainly expressed in the cytomembranes and cytoplasm of neurons. Thus, the CXCL13-CXCR5 signaling pathway may play a possible pathogenic role in IE. CXCL13 and CXCR5 may represent potential biomarkers of brain inflammation in epileptic patients.

Optimization of Neuronal-Computer Interface

DTIC Science & Technology

2009-06-23

for the inhibitory neurotransmitter gamma-aminobutyric acid ( GABA ) receptor subunit as a general marker of inhibitory neurons (Beck et al., 1993...These analyses confirmed the presence of GABA -positive neurons (Fig. 4). Fig 4: Cultures contain inhibitory neurons. Cultures were subjected to double...immunofluorescent analyses for neurofilaments (anti-NF) using monoclonal antibody SMI-32 and a polyclonal antibody directed against the GABA receptor

Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

2004-01-01

Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

L-citrulline immunostaining identifies nitric oxide production sites within neurons

NASA Technical Reports Server (NTRS)

Martinelli, G. P. T.; Friedrich, V. L. Jr; Holstein, G. R.

2002-01-01

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker. Copyright 2002 IBRO.

DNA double-strand breaks in blood lymphocytes induced by two-day 99mTc-MIBI myocardial perfusion scintigraphy.

PubMed

Rief, Matthias; Hartmann, Lisa; Geisel, Dominik; Richter, Felicitas; Brenner, Winfried; Dewey, Marc

2018-07-01

To investigate DNA double-strand breaks (DSBs) in blood lymphocytes induced by two-day 99m Tc-MIBI myocardial perfusion scintigraphy (MPS) using y-H2AX immunofluorescence microscopy and to correlate the results with 99m Tc activity in blood samples. Eleven patients who underwent two-day MPS were included. DSB blood sampling was performed before and 5min, 1h and 24h after the first and second radiotracer injections. 99m Tc activity was measured in each blood sample. For immunofluorescence microscopy, distinct foci representing DSBs were quantified in lymphocytes after staining for the phosphorylated histone variant y-H2AX. The 99m Tc-MIBI activity measured on days one and two was similar (254±25 and 258±27 MBq; p=0.594). Compared with baseline DSB foci (0.09±0.05/cell), a significant increase was found at 5min (0.19±0.04/cell) and 1h (0.18±0.04/cell) after the first injection and at 5min and 1h after the second injection (0.21±0.03 and 0.19±0.04/cell, respectively; p=0.003 for both). At 24h after the first and second injections, the number of DSB foci had returned to baseline (0.06±0.02 and 0.12±0.05/cell, respectively). 99m Tc activity levels in peripheral blood samples correlated well with DSB counts (r=0.451). DSB counts reflect 99m Tc-MIBI activity after injection for two-day MPS, and might allow individual monitoring of biological effects of cardiac nuclear imaging. • Myocardial perfusion scintigraphy using 99m Tc induces time-dependent double-strand breaks (DSBs) • γ-H2AX immunofluorescence microscopy shows DSB as an early response to radiotracer injection • Activity measurements of 99m Tc correlate well with detected DSB • DSB foci induced by 99m Tc return to baseline 24h after radiotracer injection.

DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Xu; Ptasinska, Sylwia; Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556

2013-06-10

The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

NASA Astrophysics Data System (ADS)

Han, Xu; Klas, Matej; Liu, Yueying; Sharon Stack, M.; Ptasinska, Sylwia

2013-06-01

The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models.

PubMed

Zhang, Yanke; Gao, Baobing; Xiong, Yan; Zheng, Fangshuo; Xu, Xin; Yang, Yong; Hu, Yida; Wang, Xuefeng

2017-07-01

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.

Rapid and simple method of photobleaching to reduce background autofluorescence in lung tissue sections.

PubMed

Kumar, B Santhosh; Sandhyamani, S; Nazeer, Shaiju S; Jayasree, R S

2015-02-01

Autofluorescence exhibited by tissues often interferes with immunofluorescence. Using imaging and spectral analysis, we observed remarkable reduction of autofluorescence of formalin fixed paraffin embedded tissues irradiated with light prior to incubation with immunofluorescent dyes. The technique of photobleaching offers significant improvement in the quality and specificity of immunofluorescence. This has the potential for better techniques for disease diagnosis.

Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

PubMed Central

Guttek, Karina; Hartig, Roland; Godenschweger, Frank; Roggenbuck, Dirk; Ricke, Jens; Reinhold, Dirk; Speck, Oliver

2015-01-01

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated. PMID:26176601

Production, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus).

PubMed

Tang, Xiaoqian; Liu, Fuguo; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2017-05-01

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

PubMed

Richter, D U; Jeschke, U; Bergemann, C; Makovitzky, J; Lüthen, F; Karsten, U; Briese, V

2005-01-01

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

PubMed

Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

2016-01-01

Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain.

PubMed

Billova, S; Galanopoulou, A S; Seidah, N G; Qiu, X; Kumar, U

2007-06-29

The processing of many peptides for their maturation in target tissue depends upon the presence of sorting receptor. Several previous studies have predicted that carboxypeptidase-E (CPE), prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2) may function as sorting elements for somatostatin (SST) for its maturation and processing to appropriate targets. However, nothing is currently known about whether brain, neuronal culture or even endocrine cells express SST, CPE, PC1 and PC2 and exhibit colocalization. Accordingly, in the present study using peroxidase immunohistochemistry, double-labeled indirect immunofluorescence immunohistochemistry and Western blot analysis, we mapped the distributional pattern of SST, CPE, PC1 and PC2 in different rat brain regions. Additionally, we also determined the colocalization of SST with CPE, PC1 and PC2 as well as colocalization of CPE with PC1 and PC2. The localization of SST, CPE, PC1 and PC2 reveals a distinct and region specific distribution pattern in the rat brain. Using an indirect double-label immunofluorescence method we observed selective neuron specific colocalization in a region specific manner in cortex, striatum and hippocampus. These studies provide the first evidence for colocalization between SST, CPE, PC1 and PC2 as well as CPE with PC1 and PC2. SST in cerebral cortex colocalized in pyramidal and non-pyramidal neurons with CPE, PC1 and PC2. Most importantly, in striatum and hippocampus colocalization was mostly observed selectively and preferentially in interneurons. CPE is also colocalized with PC1 and PC2 in a region specific manner. The data presented here provide a new insight into the distribution and colocalization of SST, CPE, PC1 and PC2 in rat brain. Taken together, our data anticipate the possibility that CPE, PC1 and PC2 might be potential target for the maturation of SST.

HOXB7: An Oncogenic Gene in Breast Cancer

DTIC Science & Technology

2006-05-01

novel roles for homeodomain-containing proteins include the role of human proline-rich homeodomain protein, PRH (known as Hex in studies on...immunofluorescence but not by IHC. We have now generated antibodies in chicken since the antigenicity of the human peptide may not be high in rabbits or mice since...Pandita, T. K. (2004). The role of the DNA double-strand break response network in meiosis . DNA Repair (Amst) 3, 1149-1164. Rubin, E., Mittnacht, S

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs

PubMed Central

1990-01-01

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single- strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein. PMID:2121740

Analysis of the Cellular Stress Response During Ebola Virus Infection by Immunofluorescence.

PubMed

Nelson, Emily V; Schmidt, Kristina M

2017-01-01

In this chapter, the use of immunofluorescence analysis as a tool to examine stress granule (SG) formation in Ebola virus (EBOV)-infected cells is described. The following protocol focuses on the process of inducing and analyzing the cellular stress response, including treatment of cells with inducers and inhibitors of the SG formation, and also describes EBOV infection, DNA transfection, and the usage of different cell lines.

Variations of attractors and wavelet spectra of the immunofluorescence distributions for women in the pregnant period

NASA Astrophysics Data System (ADS)

Galich, Nikolay E.

2008-07-01

Communication contains the description of the immunology data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for women in the pregnant period allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions, their bifurcation and wavelet spectra. Heterogeneity peculiarities of long-range scale immunofluorescence distributions and peculiarities of wavelet spectra allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Peculiarities of immunofluorescence for women in pregnant period are classified. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

Biostatistical analysis of quantitative immunofluorescence microscopy images.

PubMed

Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

2016-12-01

Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results. PMID:3032390

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

PubMed Central

Kato, Takamitsu A.; Suzuki, Takehiro; Dohmae, Naoshi; Takizawa, Kazuya; Nakazawa, Yuka; Genet, Matthew D.; Saotome, Mika; Hama, Michio; Nakajima, Nakako Izumi; Hazawa, Masaharu; Tomita, Masanori; Koike, Manabu; Noshiro, Katsuko; Tomiyama, Kenichi; Obara, Chizuka; Gotoh, Takaya; Ui, Ayako; Fujimori, Akira; Nakayama, Fumiaki; Sugasawa, Kaoru; Okayasu, Ryuichi; Tajima, Katsushi

2018-01-01

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism. PMID:29590107

Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

PubMed

Liang, Z-Z; Li, J; Huang, S-G

2017-03-30

The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-β1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-β-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm 2 ) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-β1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-β1 expressing macrophages varied with human chronic periapical diseases. The TGF-β1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

Immunomicrobiological monitoring of orovaccinal therapy in acute intestinal infections.

PubMed

Corridori, S; Negretti, F

1983-01-01

A double blind study in 42 patients (treated and controls) affected by acute uncomplicated salmonellosis was performed. The treatment consisted of an oral inactivated tetravaccine at the dose of 1 ampoule twice a day for 15 consecutive days. Microbiological and serological techniques (determination of intestinal secretory IgA by means of immunofluorescence, titration of copro and serum antibodies, quali-quantitative coprocultures) have shown that the administration of oral vaccine improved both the clinical and the immunomicrobiological picture in treated patients.

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia.

PubMed

Rutter, A Richard; Ma, Qing-Ping; Leveridge, Mathew; Bonnert, Timothy P

2005-11-07

Coassociation of the vanilloid transient receptor potential (Trp) ion channels, TrpV1 and TrpV2, was investigated by immunoprecipitation and immunofluorescence in transfected mammalian cell lines, rat dorsal root ganglia and spinal cord. TrpV1/TrpV2 heteromeric complexes were coimmunoprecipitated from human embryonic kidney cells and F-11 dorsal root ganglion hybridoma cells following their transient coexpression. Immunofluorescent labelling of transfected F-11 cells revealed colocalization of TrpV1 and TrpV2 at the cell surface. Immunoprecipitation from rat dorsal root ganglion lysates identified a minor population of receptor complexes composed of TrpV1/TrpV2 heteromers, consistent with a small proportion of cells double-labelled with TrpV1 and TrpV2 antibodies in rat dorsal root ganglion sections. TrpV1/TrpV2 receptor complexes may represent a functionally distinct ion channel complex that may increase the diversity observed within the Trp ion channel family.

Repolarization of hepatocytes in culture.

PubMed

Talamini, M A; Kappus, B; Hubbard, A

1997-01-01

We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

Growth and differentiation of human lens epithelial cells in vitro on matrix

NASA Technical Reports Server (NTRS)

Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

2000-01-01

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Delay, change and bifurcation of the immunofluorescence distribution attractors in health statuses diagnostics and in medical treatment

NASA Astrophysics Data System (ADS)

Galich, Nikolay E.; Filatov, Michael V.

2008-07-01

Communication contains the description of the immunology experiments and the experimental data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for healthy and unhealthy donors allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions and their bifurcation. Heterogeneity peculiarities of long-range scale immunofluorescence distributions allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Possibilities and alterations of immunofluorescence statistics in registration, diagnostics and monitoring of different diseases in various medical treatments have been demonstrated. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

PubMed Central

Mundt, E; Vakharia, V N

1996-01-01

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines. Images Fig. 2 Fig. 3 Fig. 4 PMID:8855321

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks.

PubMed

Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; Vasireddy, Raja S; El-Osta, Assam; Karagiannis, Tom C

2011-01-25

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

PubMed Central

2011-01-01

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds. PMID:21261999

[Expression and localization of transmembrane protein CMTM2 in human testis and sperm].

PubMed

Zhang, X W; Lan, K; Yang, W B; Li, Q; Zhao, Y P; Yin, H Q; Kite, B; Bai, W J; Xu, T

2017-08-18

To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system. The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction. CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction. Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

PubMed

Rybak, Paulina; Hoang, Agnieszka; Bujnowicz, Lukasz; Bernas, Tytus; Berniak, Krzysztof; Zarębski, Mirosław; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy

2016-08-02

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

An immunohistochemical study of APG-2 protein in the rat hippocampus after transient forebrain ischemia.

PubMed

Lee, Mun-Yong; Choi, Yun-Sik; Choi, Jeong-Sun; Min, Do Sik; Chun, Myung-Hoon; Kim, Ok Nyu; Lee, Sang Bok; Kim, Seong Yun

2002-01-11

The cellular localization and spatiotemporal expression pattern of APG-2 protein, a member of the heat shock protein 110 family, were investigated in the rat hippocampus after transient forebrain ischemia. The spatiotemporal patterns of immunoreactivity of both APG-2 and glial fibrillary acidic protein were very similar, indicating that reactive astrocytes express APG-2, which was confirmed by double immunofluorescence histochemistry. Colocalization of APG-2 and a neuronal marker NeuN in the neurons of the CA2 and CA3 subfields was also confirmed.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

PubMed

Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

2016-01-01

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

PubMed

Shen, S Q; Wang, R; Huang, S G

2017-03-08

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P < 0.01). There were no significant differences in CD334-SCF double-positive FB and CD31-SCF double-positive EC levels between the two periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P < 0.01). FB, EC, and MP levels were significantly high and densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs improved considerably in chronic periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

PubMed Central

Hira-Kazal, R; Shea-Simonds, P; Peacock, J L; Maher, J

2015-01-01

Anti-nuclear antibody (ANA) testing assists in the diagnosis of several immune-mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp-2) cells. However, many laboratories test for these antibodies using solid-phase assays such as enzyme-linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp-2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp-2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISANEG HEp-2POS ANA were reactive with a panel of six extractable nuclear antigens or with double-stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA-associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen. PMID:25412573

Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

PubMed Central

Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

2011-01-01

DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

PubMed

Dillinger, Stefan; Németh, Attila

2016-01-01

The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.

The Effect of Propofol on Mitochondrial Fission during Oxygen-Glucose Deprivation and Reperfusion Injury in Rat Hippocampal Neurons.

PubMed

Wang, Haibin; Zheng, Shengfa; Liu, Maodong; Jia, Changxin; Wang, Shilei; Wang, Xue; Xue, Sha; Guo, Yunliang

2016-01-01

The neuroprotective role of propofol in transient global and focal cerebral ischemia reperfusion (I/R) animal model has recently been highlighted. However, no studies have conducted to explore the relationship between mitochondrial fission/fusion and I/R injury under the intervention of propofol. Moreover, neuroprotective mechanism of propofol is yet unclear. Culturing primary hippocampal cells were subjected to oxygen-glucose deprivation and re-oxygenation (OGD/R) model, as a model of cerebral I/R in vitro. Methods CCK-8 assay was used to test the effect of propofol on cell viability. We examined the effect of propofol on mitochondrial ultrastructure and mitochondrial fission evoked by OGD/R with transmission electron microscopy and immunofluorescence assay. To investigate possible neuroprotective mechanisms, the authors then examined whether propofol could inhibit calcium-overload, calcineurin (CaN) activation and the phosphorylation of dynamin-related protein 1 (Drp1) during the period of OGD/R, as well as the combination of Drp1-ser 637 and fission 1 (Fis1) protein by immunofluorescence assay, ELISA and double-labeling immunofluorescence analysis. Finally, the expression of Drp1-ser 637 and Fis1, apoptosis inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blot. When added in culture media during OGD period, propofol (0.1μM-50μM) could alleviate neurons injury and protect mitochondrial ultrastructure, meanwhile inhibit mitochondrial fission. Furthermore, the concentration of intracellular free Ca2+, CaN activition and the phosphorylation of Drp1-ser637 were suppressed, as well as the translocation and combination of Drp1-ser 637 and Fis1. The authors also found that the expression of Cyt C, AIF, Drp1-ser637 and Fis1 were down-regulated. Notably, high dose of propofol (100μM-200μM) were confirmed to decrease the survival of neurons based on results of cell viability. Propofol could inhibit mitochondrial fission and mitochondrial apoptotic pathway evoked by OGD/R in rat hippocampal neurons, which may be via depressing calcium-overload.

The Modulation of Fibrosis in Scleroderma by 3-Deoxyglucosone

DTIC Science & Technology

2010-06-01

stained and analyzed for expression of GADD153 in the nucleus by immunofluorescence analysis using Cy3- conjugated secondary antibody . Mean...with the anti-Nox4 polyclonal antibody and Cy2-conjugated secondary antibody . Images were taken at 40 X magnification on an epi-fluorescence...GADD153 in the nucleus by immunofluorescence using a Cy3-conjugated secondary antibody . Representative images were taken at 40 X magnification on an

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

Imaging of Chromosome Dynamics in Mouse Testis Tissue by Immuno-FISH.

PubMed

Scherthan, Harry

2017-01-01

The mouse (Mus musculus) represents the central mammalian genetic model system for biomedical and developmental research. Mutant mouse models have provided important insights into chromosome dynamics during the complex meiotic differentiation program that compensates for the genome doubling at fertilization. Homologous chromosomes (homologues) undergo dynamic pairing and recombine during first meiotic prophase before they become partitioned into four haploid sets by two consecutive meiotic divisions that lack an intervening S-phase. Fluorescence in situ hybridization (FISH) has been instrumental in the visualization and imaging of the dynamic reshaping of chromosome territories and mobility during prophase I, in which meiotic telomeres were found to act as pacemakers for the chromosome pairing dance. FISH combined with immunofluorescence (IF) co-staining of nuclear proteins has been instrumental for the visualization and imaging of mammalian meiotic chromosome behavior. This chapter describes FISH and IF methods for the analysis of chromosome dynamics in nuclei of paraffin-embedded mouse testes. The techniques have proven useful for fresh and archived paraffin testis material of several mammalian species.

Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Jun; Department of Orthopaedics, Xishan People's Hospital, Wuxi, Jiangsu; Ni, Yingjie

2016-01-22

Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in themore » spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.« less

Double labeling of human leukemic cells using /sup 3/H-cytarabine and monoclonal antibody against bromodeoxyuridine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, A.; Preisler, H.D.

A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of /sup 3/H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to /sup 3/H-cytarabine. Other radioactively labeled probes can be used as well.

Regulation of the Prostate Cancer Tumor Microenvironment

DTIC Science & Technology

2017-06-01

lipopolysaccharide   (LPS)   found   in   gram -­negative   bacterial  walls   recognized   by   TLR4,   double  stranded  RNA  produced  by  many  viruses  for...W81 XWH-11-1-0260 6       We  performed  further   staining  of  infiltrating  immune  populations  in  tumors  from  30   week-­old   MyD88...immunofluorescence staining , and by qPCR, to characterize tumor-infiltrating immune populations as well as activation of the downstream NF-kB pathway and

Immunomodulatory activity of interleukin-27 in human chronic periapical diseases.

PubMed

Li, Juan; Wang, Rong; Huang, Shi-Guang

2017-01-01

This study aims to observe expression of IL-27 on different cells in periapical tissues of different types of human chronic periapical diseases. Periapical tissue specimens of 60 donors, including healthy control (n=20), periapical granuloma group (n=20) and radicular cysts group (n=20), were fixed in 10% buffered formalin, stained with hematoxylin and eosin for histopathology. Then specimens were stained with double- immuno-fluorescence assay for identification of IL-27-tryptase (mast cells, MCs), IL-27-CD14 (mononuclear phagocyte cells, MPs) and IL-27-CD31 (endothelial cells, ECs) double-positive cells in periapical tissues. The results indicated that compared with healthy control, the densities (cells/mm 2 ) of IL-27-tryptase, IL-27-CD14 and IL-27-CD31 double-positive cells were significantly increased in human chronic periapical diseases (periapical granuloma group and radicular cysts group) ( P <0.001). The density of IL-27-tryptase double positive cells in radicular cysts group was significantly higher than those in periapical granuloma group ( P <0.001). Densities of IL-27-CD14 and IL-27-CD31 double-positive cells in periapical granuloma group had no significant difference with those in radicular cysts group ( P =0.170 and 0.138, respectively). IL-27-CD14 double positive cells density achieved to peak among three cell groups in radicular cysts groups. In conclusion, IL-27 expressed in MCs, MPs and ECs of human chronic periapical diseases with different degrees. IL-27-tryptase double-positive cells may participate in pathogenic mechanism of chronic periapical diseases, especially for formation of fibrous in periapical cysts. IL-27-CD14 and IL-27-CD31 double-positive cells may participate in immunologic response to resist periapical infection, and they may play an dual role in pathogenesis and localization of periapical diseases.

Evaluation of the antiproliferative, proapoptotic, and antiangiogenic effects of a double-stranded RNA mimic complexed with polycations in an experimental mouse model of leiomyoma.

PubMed

García-Pascual, Carmen Maria; Ferrero, Hortensia; Juarez, Irene; Martínez, Jessica; Villanueva, Ana; Pozuelo-Rubio, Mercedes; Soengas, Marisol; Tormo, Damiá; Simón, Carlos; Gómez, Raúl; Pellicer, Antonio

2016-02-01

To assess the antiproliferative, proapoptotic, and antiangiogenic effects of the double-stranded RNA mimic polyinosine-polycytidylic acid (pIC) complexed with polyethylenimine [pIC(PEI)] in xenografted human leiomyomas. Heterologous leiomyoma mouse model. University-affiliated infertility center. Ovariectomized and hormone-replaced nude mice (n = 16) who received human leiomyoma fragment transplantation. Leiomyoma fragments placed in the peritoneum of 5-week-old nude female mice and treated with the vehicle (n = 8) or 0.6 mg/kg [pIC(PEI)] (n = 8) for 4 weeks. The size of the leiomyoma implants, and cellular proliferation (Ki67), vascularization (PECAM), and apoptosis (OH-ends) assessed by quantitative immunohistochemical/immunofluorescent analysis of the recovered implants. No significant differences were observed in the size of the leiomyoma implants between groups. Vascularization and proliferation were significantly decreased, and apoptosis was increased in the [pIC(PEI)]-treated group versus control. We hypothesize that the antiangiogenic and apoptotic effects exerted by [pIC(PEI)] might lead to a decrease in lesion size in this animal model if the compound is administered for longer periods of time. This study provides promising data on [pIC(PEI)] as a potential novel therapeutic agent against human leiomyoma. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Immunoglobulin E (IgE) and IgE-containing cells in human gastrointestinal fluids and tissues.

PubMed Central

Brown, W R; Borthistle, B K; Chen, S T

1975-01-01

Human gastric, small intestinal, colonic and rectal mucosae were examined for IgE-containing cells by single- and double-antibody immunofluorescence techniques, and IgE in intesinal fluids was measured by a double-antibody radioimmunoassay. IgE-containing cells were identified in all tissue specimens and comprised about 2% of all immunoglobulin-containing cells. Although less numerous than cells containing IgA, IgM or IgG, they were remarkably numerous in relation to the concentration of IgE in serum (about 0-001% of total immunoglobulin). IgE immunocytes were significantly more numerous in stomach and proximal small bowel than in colon and rectum, and were very numerous at bases of gastric and duodenal peptic ulcers. Measurable IgE was found in seventy-eight of eighty-five (92%) intestinal fluids. Sucrose gradient ultracentrifugation analysis of four of the fluids revealed that the immunologically reactive IgE was largely in fractions corresponding to molecules of lower molecular weight than that of albumin, which suggests that IgE in gut contents is degraded by proteolytic enzymes. The presence of IgE-forming cells in gastrointestinal tissues, and IgE or a fragment of IgE in intestinal fluids, suggests that IgE antibodies are available for participation in local reaginic-type reactions in the gut. Images FIG. 1 PMID:813925

High sensitive detection of double-stranded DNA autoantibodies by a modified Crithidia luciliae immunofluorescence test.

PubMed

Conrad, Karsten; Ittenson, Annelore; Reinhold, Dirk; Fischer, Richard; Roggenbuck, Dirk; Büttner, Thomas; Bosselmann, Hans-Peter; Steinbach, Jörg; Schössler, Werner

2009-09-01

Anti-double-stranded (ds)DNA antibodies are serological markers of systemic lupus erythematosus (SLE). Of all anti-dsDNA antibody detection methods, the Crithidia luciliae immunofluorescence test (CLIFT) is thought to have the highest specificity for SLE. However, the clinical application is hampered by the low diagnostic sensitivity. A CLIFT with modified assay buffer (mCLIFT) was developed and compared with conventional CLIFT, using sera from 110 patients with SLE, 89 anti-dsDNA ELISA-positive patients with other diseases (non-SLE group A), 157 non-SLE patients with undetectable anti-dsDNA antibodies by ELISA (non-SLE group B), 77 disease controls (non-SLE group C), and 50 healthy blood donors. Out of the 110 anti-dsDNA antibody ELISA-positive SLE patients, 84 (76.4%) demonstrated a positive kinetoplast staining, using the mCLIFT, compared to only 42.3%, using the conventional CLIFT. The diagnostic specificity of mCLIFT was 100% with healthy blood donors and 98.1% with the non-SLE group C (anti-nuclear antibodies negative; no signs or symptoms of an autoimmune disease) included. In the non-SLE groups A and B with various other autoimmune diseases or symptoms of a possible autoimmune disease, positive mCLIFT results were obtained in 33.7% and 3.2%, respectively. In conclusion, by modification of the assay buffer, a significant increase in sensitivity of the CLIFT could be observed while retaining the high specificity for SLE. Further investigation is required to check whether the CLIFT-positive non-SLE patients develop SLE and whether anti-dsDNA antibodies detected by the mCLIFT represent a pathogenetic and diagnostic subgroup of autoantibodies that may improve the early diagnosis of SLE or SLE-overlap syndromes.

Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia.

PubMed

Friedmacher, Florian; Fujiwara, Naho; Hofmann, Alejandro D; Takahashi, Hiromizu; Alvarez, Luis A J; Gosemann, Jan-Hendrik; Puri, Prem

2014-06-01

Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH. Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo, and Nitrofen+ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining. Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo (0.31±0.02 vs. 0.08±0.01; P<0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen+ATRA compared to Nitrofen+Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen+ATRA compared to Nitrofen+Placebo. Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development. Copyright © 2014 Elsevier Inc. All rights reserved.

Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

PubMed

Fontaine, Romain; Affaticati, Pierre; Yamamoto, Kei; Jolly, Cécile; Bureau, Charlotte; Baloche, Sylvie; Gonnet, Françoise; Vernier, Philippe; Dufour, Sylvie; Pasqualini, Catherine

2013-02-01

In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.

TAAR1 induces a disturbed GSK3β phosphorylation in recurrent miscarriages through the ODC

PubMed Central

Stavrou, Stavroula; Gratz, Michael; Tremmel, Eileen; Kuhn, Christina; Hofmann, Simone; Heidegger, Helene; Peryanova, Mina; Hermelink, Kerstin; Hutter, Stefan; Toth, Bettina; Mayr, Doris; Mahner, Sven; Jeschke, Udo; Vattai, Aurelia

2018-01-01

Objectives Thyroid hormones play an important role in the maintenance of pregnancy. Their derivates, endogenous amines, act via binding to the trace amine-associated receptor (TAAR1). The aim of our study was to analyse the regulation of TAAR1, serine/threonine kinase (pGSK3β) and ornithine decarboxylase (ODC) in placentas of healthy pregnancies, spontaneous (SM) and recurrent miscarriages (RM) and to investigate the influence of thyroid hormone derivates on TAAR1 expression in trophoblast model cells in vitro. Methods Patients with SM (n = 15) and RM (n = 15) were compared with patients with healthy pregnancies (n = 15) (pregnancy weeks 7–13 each). Immunohistochemistry was applied to analyse placental TAAR1, pGSK3β and ODC expression. Protein expression of the receptors after stimulation with T3, T1AM and RO5203548 in BeWo trophoblast model cells was determined via Western blot. Double-immunofluorescence was used to determine placental expression of TAAR1 and ODC. Results Levels of TAAR1, pGSK3β and ODC were higher in placentas of RM in comparison to healthy controls. Stimulation of BeWo cells with T3, T1AM and RO5203548 significantly increased TAAR1 expression. ODC expression in BeWo cells was upregulated through T3. Via double-immunofluorescence, TAAR1 and ODC-positive EVT could be detected. Conclusions Upregulation of placental TAAR1 may indicate an increased decarboxylation of thyroid hormones in miscarriages. Patients with RM may have a lack of T3 through an enhanced transformation of T3 into T1AM induced by the ODC. Future investigations could be carried out to analyse what role a prophylactic T3 substitution plays for patients. PMID:29472377

Doublecortin is expressed in trigeminal motoneurons that innervate the velar musculature of lampreys: considerations on the evolution and development of the trigeminal system.

PubMed

Barreiro-Iglesias, Antón; Romaus-Sanjurjo, Daniel; Senra-Martínez, Pablo; Anadón, Ramón; Rodicio, María Celina

2011-01-01

Studies in lampreys have revealed interesting aspects of the evolution of the trigeminal system and the jaw. In the present study, we found a marker that distinguishes subpopulations of trigeminal motoneurons innervating two different kinds of oropharyngeal muscles. Immunofluorescence with an antibody against doublecortin (DCX; a neuron-specific phosphoprotein) enabled identification of the trigeminal motoneurons that innervate the velar musculature of larval and recently transformed sea lampreys. DCX-immunoreactive (-ir) motoneurons were observed in the rostro-lateral part of the trigeminal motor nucleus of these animals, but not in lampreys 1 month or more after metamorphosis. Combined double DCX/tubulin and serotonin/tubulin immunofluorescence and tract-tracing experiments with neurobiotin (NB) were also performed in larvae for further characterization of this system. Rich innervation by DCX-ir fibers was observed on the muscle fibers of the velum but not on the upper lip or lower lip muscles, which were innervated by tubulin-ir/DCX-negative fibers. No double-labelled DCX-ir motoneurons were observed in experiments in which the tracer NB was applied to the upper lip. Innervation of velar muscles by serotonergic fibers is also reported. The present results indicate that development of the trigeminal motoneurons innervating the velum differs from that of the trigeminal motoneurons innervating the lips, which is probably related to the dramatic regression of the velum during metamorphosis. The absence of data on a similar subsystem in the trigeminal motor nucleus of gnathostomes suggests that they may be lamprey-specific motoneurons. These results provide support for the "heterotopic theory" of jaw evolution and are inconsistent with the theories of a velar origin for the gnathostome jaw. © 2011 Wiley Periodicals, Inc.

Tumor necrosis factor and its receptors in the neuroretina and retinal vasculature after ischemia-reperfusion injury in the pig retina

PubMed Central

Gesslein, Bodil; Håkansson, Gisela; Gustafsson, Lotta; Ekström, Per

2010-01-01

Purpose Numerous studies have been performed aimed at limiting the extent of retinal injury after ischemia, but there is still no effective pharmacological treatment available. The aim of the present study was to examine the role of tumor necrosis factor (TNF)α and its receptors (TNF-R1 and TNF-R2), especially considering the neuroretina and the retinal vasculature since the retinal blood vessels are key organs in circulatory failure. Methods Retinal ischemia was induced in pigs by elevating the intraocular pressure to 80 mmHg in one eye, while the other eye served as a control (sham-operated). One hour of ischemia was followed by 5 or 12 h of reperfusion. Retinal circulation was examined in vivo by fundus imaging and fluorescein angiography. TNF-α levels were measured in the vitreous using an angiogenesis antibody array test. The presence and amounts of TNF-α, TNF-R1, and TNF-R2 were investigated in the neuroretina and in the retinal blood vessels, using immunofluorescence staining and real-time PCR techniques. Results Fundus imaging showed obstructed blood flow when ischemia was induced, and reperfusion was clearly visualized using fluorescein angiography. Ischemia resulted in elevated levels of TNF-α protein in the vitreous and TNF-α mRNA in the neuroretina. TNF-α immunofluorescence staining was localized to the Müller cells and the outer plexiform layer of the neuroretina. The expression of TNF-R1 and TNF-R2 mRNA was increased in both the neuroretina and retinal arteries following ischemia-reperfusion. Immunofluorescence double staining for TNF-R1 and either smooth muscle actin or 4',6-diamidino-2-phenylindole (DAPI) indicated expression in the cell membranes of the vascular smooth muscle cells. Double staining with TNF-R1 and calbindin showed localization to the horizontal cells in the outer plexiform layer of the neuroretina. Conclusions Retinal ischemia results in increased expression of TNF-α and its receptors (TNF-R1 and TNF-R2). Cellular signaling pathways involving TNF may be important in the development of retinal injury following ischemia and thus an interesting target for future development of pharmacological therapeutics. PMID:21152396

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

PubMed

Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

2013-01-01

To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7weeks of radiotherapy.

PubMed

Qvarnström, Fredrik; Simonsson, Martin; Nyman, Jan; Hermansson, Ingegerd; Book, Majlis; Johansson, Karl-Axel; Turesson, Ingela

2017-01-01

Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05-1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. HRS in keratinocytes was evidenced by the dose-response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose-response relationships. These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

WE-E-BRE-03: Biological Validation of a Novel High-Throughput Irradiator for Predictive Radiation Sensitivity Bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, TL; Martin, JA; Shepard, AJ

2014-06-15

Purpose: The large dose-response variation in both tumor and normal cells between individual patients has led to the recent implementation of predictive bioassays of patient-specific radiation sensitivity in order to personalize radiation therapy. This exciting new clinical paradigm has led us to develop a novel high-throughput, variable dose-rate irradiator to accompany these efforts. Here we present the biological validation of this irradiator through the use of human cells as a relative dosimeter assessed by two metrics, DNA double-strand break repair pathway modulation and intercellular reactive oxygen species production. Methods: Immortalized human tonsilar epithelial cells were cultured in 96-well micro titermore » plates and irradiated in groups of eight wells to absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy. High-throughput immunofluorescent microscopy was used to detect γH2AX, a DNA double-strand break repair mechanism recruiter. The same analysis was performed with the cells stained with CM-H2DCFDA that produces a fluorescent adduct when exposed to reactive oxygen species during the irradiation cycle. Results: Irradiations of the immortalized human tonsilar epithelial cells at absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy produced excellent linearity in γH2AX and CM-H2DCFDA with R2 values of 0.9939 and 0.9595 respectively. Single cell gel electrophoresis experimentation for the detection of physical DNA double-strand breaks in ongoing. Conclusions: This work indicates significant potential for our high-throughput variable dose rate irradiator for patient-specific predictive radiation sensitivity bioassays. This irradiator provides a powerful tool by increasing the efficiency and number of assay techniques available to help personalize radiation therapy.« less

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

PubMed Central

Johnson-Tidey, R. R.; McGregor, J. L.; Taylor, P. R.; Poston, R. N.

1994-01-01

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7513951

Follistatin-like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Zimmer, Julia; Friedmacher, Florian; Puri, Prem

2017-05-01

Pulmonary hypoplasia (PH), characterized by incomplete alveolar development, remains a major therapeutic challenge associated with congenital diaphragmatic hernia (CDH). Follistatin-like 1 (Fstl1) is a crucial regulator of alveolar formation and maturation, which is strongly expressed in distal airway epithelium. Fstl1-deficient mice exhibit reduced airspaces, impaired alveolar epithelial cell differentiation, and insufficient production of surfactant proteins similar to PH in human CDH. We hypothesized that pulmonary Fstl1 expression is decreased during alveolarization in the nitrofen-induced CDH model. Timed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 and divided into control-/nitrofen-exposed specimens. Alveolarization was assessed using morphometric analysis techniques. Pulmonary gene expression of Fstl1 was determined by qRT-PCR. Immunofluorescence-double-staining for Fstl1 and alveolar epithelial marker surfactant protein C (SP-C) was performed to evaluate protein expression/localization. Radial alveolar count was significantly reduced in hypoplastic lungs of nitrofen-exposed fetuses with significant down regulation of Fstl1 mRNA expression on D18 and D21 compared to controls. Confocal-laser-scanning-microscopy revealed strikingly diminished Fstl1 immunofluorescence and SP-C expression in distal alveolar epithelium of nitrofen-exposed fetuses with CDH-associated PH on D18 and D21 compared to controls. Decreased expression of Fstl1 in alveolar epithelium may disrupt alveolarization and pulmonary surfactant production, thus contributing to the development of PH in the nitrofen-induced CDH model. 2b (Centre for Evidence-Based Medicine, Oxford). Copyright © 2017 Elsevier Inc. All rights reserved.

Magneto immunofluorescence assay for diagnosis of celiac disease.

PubMed

Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

2013-10-10

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit. Copyright © 2013 Elsevier B.V. All rights reserved.

[Silencing MSH3 expression enhances cisplatin sensitivity of human tongue cancer cells].

PubMed

Fan, Xiao-Sheng; Cao, Fang-Yun; Li, Kuang-Zheng

2016-08-20

To explore the effect of MSH3 knock-down on sensitivity of tongue cancer cells to cisplatin. Three small interfering RNA (siRNA) fragments targeting MSH3 CDS region were synthesized and transfected into CAL27 cells via Lipofectamine. Real-time PCR and Western blotting were used to assess the efficiency of MSH3 silencing. MTS, apoptosis staining and cell immunofluorescence assay were used to examine the cisplatin sensitivity, apoptosis and DNA repair of transfected CAL27 cells. s One of the 3 siRNAs was found to significantly reduce the expression of MSH3 protein in CAL27 cells (P<0.05). MTS assay showed that MSH3 silencing resulted in an significant reduction of IC50 of cisplatin from 21.32 to 13.95 µmol/L (P<0.05) and increased the apoptotic index of the exposed cells from 4.23∓1.27 to 11.32∓1.82 (P<0.05). Immunofluorescence assay demonstrated that silencing MSH3 markedly reduced the number of γ-H2AX foci. Silencing MSH3 can significantly increase cisplatin sensitivity of tongue cancer cells, the mechanism of which involves mainly attenuation of repair of DNA double-strand damage in the cells.

Deficiency of endothelial CXCR4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice.

PubMed

Noels, Heidi; Zhou, Baixue; Tilstam, Pathricia V; Theelen, Wendy; Li, Xiaofeng; Pawig, Lukas; Schmitz, Corinna; Akhtar, Shamima; Simsekyilmaz, Sakine; Shagdarsuren, Erdenechimeg; Schober, Andreas; Adams, Ralf H; Bernhagen, Jürgen; Liehn, Elisa A; Döring, Yvonne; Weber, Christian

2014-06-01

The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. β-Galactosidase staining using bone marrow x kinase (Bmx)-CreER(T2) reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER(T2+) compared with Bmx-CreER(T2-) Cxcr4-floxed apolipoprotein E-deficient (Apoe(-/-)) mice (referred to as Cxcr4(EC-KO)ApoE(-/-) and Cxcr4(EC-WT) ApoE(-/-), respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4(EC-KO)ApoE(-/-) mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4(EC-KO)ApoE(-/-) carotids compared with Cxcr4(EC-WT)ApoE(-/-) controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1(+)Flk1(+)Cd31(+) and of Lin(-)Sca1(+) progenitors in Cxcr4(EC-KO) ApoE(-/-) mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4(EC-KO)ApoE(-/-) mice. Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1(+)Flk1(+)Cd31(+) cells, often referred to as circulating endothelial progenitor cells. © 2014 American Heart Association, Inc.

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat.

PubMed

Wang, S G; Tsao, D D; Vanderpool, K G; Yasumura, T; Rash, J E; Nagy, J I

2017-06-01

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Huang, Yongping; Xie, Mingquan; Luo, Xiaochun; Li, Wenmei; Wang, Jihua

2015-05-30

The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. The cut-off level of detection of the kit was 25 parasites/μl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

PubMed

Poulsen, Nicklas N; Pedersen, Morten E; Østergaard, Jesper; Petersen, Nickolaj J; Nielsen, Christoffer T; Heegaard, Niels H H; Jensen, Henrik

2016-09-20

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

PubMed Central

Zimmerman, Sandra G; Peters, Nathaniel C; Altaras, Ariel E; Berg, Celeste A

2014-01-01

In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase–conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform. PMID:24113787

DNA Double-strand Breaks Induced byFractionated Neutron Beam Irradiation for Boron Neutron Capture Therapy.

PubMed

Kinashi, Yuko; Yokomizo, Natsuya; Takahashi, Sentaro

2017-04-01

To use the 53BP1 foci assay to detect DNA double-strand breaks induced by fractionated neutron beam irradiation of normal cells. The Kyoto University Research Reactor heavy-water facility and gamma-ray irradiation system were used as experimental radiation sources. After fixation of Chinese Hamster Ovary cells with 3.6% formalin, immunofluorescence staining was performed. Number and size of foci were analyzed using ImageJ software. Fractionated neutron irradiation induced 25% fewer 53BP1 foci than single irradiation at the same dose. By contrast, gamma irradiation induced 30% fewer 53BP1 foci than single irradiation at the same dose. Fractionated neutron irradiation induced larger foci than gamma irradiation, raising the possibility that persistent unrepaired DNA damage was amplified due to the high linear energy transfer component in the neutron beam. Unrepaired cluster DNA damage was more prevalent after fractionated neutron irradiation than after gamma irradiation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[Establishment and characterization of a new carcinoma cell line from uterine cervix of Uyghur women].

PubMed

Zhang, Lu; Aerziguli, Tursun; Guzalnur, Abliz

2012-04-01

To establish a uterine cervical carcinoma cell line of Uyghur ethnical background and to evaluate the related biological characteristics for future biomedical investigations of diseases in the Uyghur population. Poorly-differentiated squamous cell carcinoma specimens of Uyghur patients were obtained and cultured in vitro by enzymatic digestion method, followed by continuous passaging to reach a stable growth determined by cell viability and growth curve. Morphological study, cell cycling and chromosomal analysis were performed. Tumorigenesis study was conducted by inoculation of nude mice. Biomarker (CK17, CD44, Ki-67, CK14 and vimentin) expression was detected by immunofluorescence and immunocytochemical techniques. A cervical carcinoma cell line was successfully established and maintained for 12 months through 70 passages. The cell line had a stable growth with a population doubling time of 51.9 h. Flask method and double agar-agar assay showed that the cell line had colony-forming rates of 32.5% and 15.6%, respectively. Ultrastructural evaluation demonstrated numerous cell surface protrusions or microvilli, a large number of rod-shape structures in cytoplasm, typical desmosomes and nuclear atypia. Chromosomal analysis revealed human karyotype with the number of chromosomes per cell varying from 32 - 97 with a majority of 54 - 86 (60.3%). Xenogeneic tumors formed in nude mice showed histological structures identical to those of the primary tumor. The cells had high expression of CK17, CD44, Ki-67 and vimentin but no CK14 expression. A cervical carcinoma cell line from a female Uyghur patient is successfully established. The cell line has the characteristics of human cervical squamous cell carcinoma, and it is stable with maintaining the characteristic biological and morphological features in vitro for more than 12 months, therefore, qualified as a stable cell line for further biomedical research.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma.

PubMed

Sun, Xi-Tai; Yuan, Xian-Wen; Zhu, Hai-Tao; Deng, Zheng-Ming; Yu, De-Cai; Zhou, Xiang; Ding, Yi-Tao

2012-09-21

To investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) in the angiogenesis of hepatocellular carcinoma (HCC). The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein (GFP) + bone marrow cells. The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting. Serum and tissue levels of vascular endothelial growth factor (VEGF) and colony-stimulating factor (CSF) were quantified by enzyme-linked immunosorbent assay. The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction. The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry. The proportion of EPCs in vessels was then calculated. The HCC model was successful established. The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively. These values are much higher than in the sham-operation group (0.11% ± 0.13%, 0.05% ± 0.11%, n = 9) at 14 d after modeling. At 21 d, the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%, 0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%, 0.12% ± 0.11% in control. Compared to the transient increase observed in controls, the higher level of circulating EPCs were induced by HCC. In addition, the level of serum VEGF and CSF increased gradually in HCC, reaching its peak 14 d after modeling, then slowly decreased. Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels. Under fluorescence microscopy, the bone-marrow (BM)-derived cells labeled with GFP were concentrated in the same area. The relative levels of CD133 and CD34 gene expression were elevated in tumors, around 5.0 and 3.8 times that of the tumor free area. In frozen liver sections from HCC mice, cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies. In tumor tissue, the double-positive cells were incorporated into vessel walls. In immunofluorescent staining. These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells (VECs) come partly from BM-derived EPCs. The proportion of GFP CD31 double positive VECs (out of all VECs) on day 21 was around 35.3% ± 21.2%. This is much higher than the value recorded on day 7 group (17.1% ± 8.9%). The expression of intercellular adhesion molecule 1, vascular adhesion molecule 1, and VEGF was higher in tumor areas than in tumor-free tissues. Mobilized EPCs were found to participate in tumor vasculogenesis of HCC. Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-β activity.

PubMed

García del Caño, Gontzal; Aretxabala, Xabier; González-Burguera, Imanol; Montaña, Mario; López de Jesús, Maider; Barrondo, Sergio; Barrio, Ramón J; Sampedro, Carmen; Goicolea, M Arantzazu; Sallés, Joan

2015-03-01

In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus. © 2014 International Society for Neurochemistry.

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

PubMed

Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

2012-01-01

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Downregulation of PI3K/Akt/mTOR signaling pathway in curcumin-induced autophagy in APP/PS1 double transgenic mice.

PubMed

Wang, Chen; Zhang, Xiong; Teng, Zhipeng; Zhang, Tong; Li, Yu

2014-10-05

Autophagy is a lysosomal degradation pathway, which is essential for cell survival, proliferation, differentiation and homeostasis. It is well known that beta-amyloid (Aβ) aggregation is one of key characteristics for Alzheimer's disease (AD), which triggers a complex pathological cascade, leading to neurodegeneration. Recent studies have shown that Aβ peptide is generated from amyloid β precursor protein (APP) during autophagic turnover of APP-rich organelles by autophagy. Aβ generation during normal autophagy is subsequently degraded by lysosomes. Curcumin, a nature plant extraction, has been reported to inhibit the generation and deposition of Aβ; however, the underlying mechanisms are not fully understood yet. In the present study, we reported that curcumin treatment not only attenuated cognitive impairment detected by Morris water maze test, but also inhibited the generation of Aβ investigated by immunohistochemistry in APP/PS1 double transgenic AD mice. Moreover, curcumin induced autophagy in the mice, evidenced by LC3 immunofluorescence analysis and western blot assays on LC3. Furthermore, we found that curcumin significantly decreased the expression of Phosphatidylinositol 3-Kinase (PI3K), phosphorylated Akt and rapamycin (mTOR) at protein levels, respectively. Taken together, our data suggests that curcumin inhibits Aβ generation and induces of autophagy by downregulating PI3K/Akt/mTOR signaling pathway, and further shows a neuroprotective effect. Meanwhile curcumin might be a candidate neuroprotective agent for AD patients treatment by inducing autophagy. Copyright © 2014 Elsevier B.V. All rights reserved.

Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment

PubMed Central

Abdisalaam, Salim; Davis, Anthony J.; Chen, David J.; Alexandrakis, George

2014-01-01

A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA–PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA–PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA–PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments. PMID:24137007

Autophagic activity in BC3H1 cells exposed to yessotoxin.

PubMed

Korsnes, Mónica Suárez; Kolstad, Hilde; Kleiveland, Charlotte Ramstad; Korsnes, Reinert; Ørmen, Elin

2016-04-01

The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Increased expression of interleukin-21 along colorectal adenoma-carcinoma sequence and its predicating significance in patients with sporadic colorectal cancer.

PubMed

Cui, Guanglin; Yuan, Aping; Zhu, Li; Florholmen, Jon; Goll, Rasmus

2017-10-01

The role and significance of interleukin (IL)-21 in the development of sporadic CRC have not been well defined. The aim of this study is therefore to investigate the dynamics of the IL-21 along colorectal adenoma-carcinoma sequence and to evaluate the impact of IL-21 on clinicopathological parameters and CRC prognosis. The real-time PCR results showed that the level of IL-21 in adenomas (n=50) and sporadic CRC (n=50) were significantly higher than that in normal controls (n=18), which were predominately observed in the adenoma/CRC stroma. Analysis revealed that IL-21 level was correlated with the overall survival time in CRC patients. Double immunofluorescence observations confirmed that IL-21 positive cells were mostly natural killer cells and T lymphocytes in the tumor stroma. These results indicate that significant increased IL-21 expression present within the adenoma/CRC microenvironment might have a potential predicating significance for survival time in patients with CRC. Copyright © 2017 Elsevier Inc. All rights reserved.

Neurofibromin Regulates Seizure Attacks in the Rat Pilocarpine-Induced Model of Epilepsy.

PubMed

Ren, Min; Li, Kunyi; Wang, Dan; Guo, Jiamei; Li, Jing; Yang, Guang; Long, Xianghua; Shen, Wenjing; Hu, Rong; Wang, Xuefeng; Zeng, Kebin

2016-11-01

Studies have shown that neurofibromin (NF1) restricts GABA release at inhibitory synapses and regulates dendritic spine formation, which may play an important role in temporal lobe epilepsy (TLE). NF1 expression was detected by double-label immunofluorescence, immunohistochemistry, and western blot analysis in the brains of pilocarpine-induced epilepsy model rats at 6 h, 24 h, 72 h, 7 days, 14 days, 30 days, and 60 days after kindling. NF1 was localized primarily in the nucleus and cytoplasm of neurons. NF1 protein levels significantly increased in the chronic phase (from 7 days until 60 days) in this epileptic rat model. After NF1 expression was knocked down by specific siRNA, the effects of kindling with pilocarpine were evaluated on the 7th day after kindling. The onset latencies of pilocarpine-induced seizures were elevated, and the seizure frequency and duration were reduced in these rats. Our study demonstrates that NF1 promoted seizure attacks in rats with pilocarpine-induced epilepsy.

Immunomodulatory activity of interleukin-27 in human chronic periapical diseases

PubMed Central

Li, Juan; Wang, Rong; Huang, Shi-Guang

2017-01-01

This study aims to observe expression of IL-27 on different cells in periapical tissues of different types of human chronic periapical diseases. Periapical tissue specimens of 60 donors, including healthy control (n=20), periapical granuloma group (n=20) and radicular cysts group (n=20), were fixed in 10% buffered formalin, stained with hematoxylin and eosin for histopathology. Then specimens were stained with double- immuno-fluorescence assay for identification of IL-27-tryptase (mast cells, MCs), IL-27-CD14 (mononuclear phagocyte cells, MPs) and IL-27-CD31 (endothelial cells, ECs) double-positive cells in periapical tissues. The results indicated that compared with healthy control, the densities (cells/mm2) of IL-27-tryptase, IL-27-CD14 and IL-27-CD31 double-positive cells were significantly increased in human chronic periapical diseases (periapical granuloma group and radicular cysts group) (P<0.001). The density of IL-27-tryptase double positive cells in radicular cysts group was significantly higher than those in periapical granuloma group (P<0.001). Densities of IL-27-CD14 and IL-27-CD31 double-positive cells in periapical granuloma group had no significant difference with those in radicular cysts group (P=0.170 and 0.138, respectively). IL-27-CD14 double positive cells density achieved to peak among three cell groups in radicular cysts groups. In conclusion, IL-27 expressed in MCs, MPs and ECs of human chronic periapical diseases with different degrees. IL-27-tryptase double-positive cells may participate in pathogenic mechanism of chronic periapical diseases, especially for formation of fibrous in periapical cysts. IL-27-CD14 and IL-27-CD31 double-positive cells may participate in immunologic response to resist periapical infection, and they may play an dual role in pathogenesis and localization of periapical diseases. PMID:28386371

Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

Characterizing invading glioma cells based on IDH1-R132H and Ki-67 immunofluorescence.

PubMed

Sabit, Hemragul; Nakada, Mitsutoshi; Furuta, Takuya; Watanabe, Takuya; Hayashi, Yutaka; Sato, Hiroshi; Kato, Yukinari; Hamada, Jun-ichiro

2014-10-01

Glioma, the most common primary brain tumor, is characterized by proliferative-invasive growth. However, the detailed biological characteristics of invading glioma cells remain to be elucidated. A monoclonal antibody (clone HMab-1) that specifically and sensitively recognizes the isocitrate dehydrogenase-1 (IDH1) protein carrying the R132H mutation can identify invading glioma cells by immunostaining. To investigate the degree of invasion in gliomas of distinct grades and the proliferative capacity of the invading cells, immunofluorescent staining was conducted using antibodies against IDH1-R132H and Ki-67 on 11 surgical and autopsy specimens of the tumor core and the invading area. Higher numbers of IDH1-R132H-positive cells in the invading area correlated with a higher tumor grade. Double staining for IDH1-R132H and Ki-67 demonstrated that most invading cells that expressed IDH1-R132H were not stained by the Ki-67 antibody, and the ratio of Ki-67-positive cells among IDH1-R132H-positive cells was significantly lower in the invasion area than in the tumor core in all grades of glioma. These data suggest that higher grade gliomas have a greater invasive potential and that invading cells possess low proliferative capacity.

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization.

PubMed

Oglesbee, M; Jackwood, D; Perrine, K; Axthelm, M; Krakowka, S; Rice, J

1986-11-01

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

Deletion of the TNFAIP3/A20 gene detected by FICTION analysis in classical Hodgkin lymphoma

PubMed Central

2012-01-01

Background The TNFAIP3 gene, which encodes a ubiquitin-modifying enzyme (A20) involved in the negative regulation of NF-κB signaling, is frequently inactivated by gene deletions/mutations in a variety of B-cell malignancies. However, the detection of this in primary Hodgkin lymphoma (HL) specimens is hampered by the scarcity of Hodgkin Reed-Sternberg (HR-S) cells even after enrichment by micro-dissection. Methods We used anti-CD30 immunofluorescence with fluorescence in-situ hybridization (FISH) to evaluate the relative number of TNFAIP3/CEP6 double-positive signals in CD30-positive cells. Results From a total of 47 primary classical Hodgkin lymphoma (cHL) specimens, 44 were evaluable. We found that the relative numbers of TNFAIP3/CD30 cells were distributed among three groups, corresponding to those having homozygous (11%), heterozygous (32%), and no (57%) deletions in TNFAIP3. This shows that TNFAIP3 deletions could be sensitively detected using our chosen methods. Conclusions Comparing the results with mutation analysis, TNFAIP3 inactivation was shown to have escaped detection in many samples with homozygous deletions. This suggests that TNFAIP3 inactivation in primary cHL specimens might be more frequent than previously reported. PMID:23039325

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

PubMed

Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

2017-11-27

Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

An expert protocol for immunofluorescent detection of calcium channels in tsA-201 cells.

PubMed

Koch, Peter; Herzig, Stefan; Matthes, Jan

Pore-forming subunits of voltage gated calcium channels (VGCC) are large membrane proteins (260kDa) containing 24 transmembrane domains. Despite transfection with viral promoter driven vectors, biochemical analysis of VGCC is often hampered by rather low expression levels in heterologous systems rendering VGCC challenging targets. Especially in immunofluorescent detection, calcium channels are demanding proteins. We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28°C or 37°C and immunostaining) to address the L-type calcium-channel pore Ca v 1.2 in an immunofluorescent approach. We performed immunocytochemical analysis of Ca v 1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Ca v 1.2 protein and a correct Ca v 1.2 expression pattern in polygonal shaped cells already 12h after transfection. A sequence of elaborated protocol modifications allows subcellular localization analysis of Ca v 1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels. Copyright © 2016 Elsevier Inc. All rights reserved.

[Fractal research of neurite growth in immunofluorescent images].

PubMed

Tang, Min; Wang, Huinan

2008-12-01

Fractal dimension has been widely used in medical images processing and analysis. The neurite growth of cultured dorsal root ganglion (DRG) was detected by fluorescent immunocytochemistry treated with nerve regeneration factor (0.1, 0.5, 2.0 mg/L). A novel method based on triangular prism surface area (TPSA) was introduced and adopted to calculate the fractal dimension of the two-dimensional immunofluorescent images. Experimental results demonstrate that this method is easy to understand and convenient to operate, and the quantititve results are concordant with the observational findings under microscope. This method can be guidelines for analyzing and deciding experimental results.

Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus.

PubMed

Ng, Patricia P L; Thng, Steven T G; Mohamed, Khatija; Tan, Suat Hoon

2005-11-01

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.

Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype.

PubMed

Kristiansen, G; Rose, M; Geisler, C; Fritzsche, F R; Gerhardt, J; Lüke, C; Ladhoff, A-M; Knüchel, R; Dietel, M; Moch, H; Varga, Z; Theurillat, J-P; Gorr, T A; Dahl, E

2010-06-08

We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

PubMed

Krause, C; Ens, K; Fechner, K; Voigt, J; Fraune, J; Rohwäder, E; Hahn, M; Danckwardt, M; Feirer, C; Barth, E; Martinetz, T; Stöcker, W

2015-04-01

Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

PubMed

Spagnolo, Daniel M; Al-Kofahi, Yousef; Zhu, Peihong; Lezon, Timothy R; Gough, Albert; Stern, Andrew M; Lee, Adrian V; Ginty, Fiona; Sarachan, Brion; Taylor, D Lansing; Chennubhotla, S Chakra

2017-11-01

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR . ©2017 American Association for Cancer Research.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Colocalization of numerous immunoreactivities in endocrine cells of the chicken proventriculus at hatching.

PubMed

Martínez, A; Buchan, A M; López, J; Sesma, P

2000-05-01

The colocalization of regulatory peptide immunoreactivities in endocrine cells of the chicken proventriculus at hatching has been investigated using the avidin-biotin technique in serial sections and double immunofluorescence in the same section for light microscopy, and double immunogold staining for electron microscopy. In addition to the eight immunoreactivities previously described in this organ, cells immunoreactive for peptide histidine isoleucine (PHI), peptide gene product 9.5 (PGP), and the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) were observed. All the cells immunoreactive to glucagon were also immunostained by the PHI antiserum. In addition, all the glucagon-like peptide 1, avian pancreatic polypeptide, and some of the neurotensin-like cells costored also glucagon- and PHI-immunoreactive substances. PGP- and PAM-immunoreactivities were also found in the glucagon-positive cells. A small proportion of the somatostatin-containing cells were positive for PHI but not for other regulatory peptides. These results could suggest either the existence of a very complex regulatory system or that the endocrine system of the newborn chickens is not yet fully developed.

Coexpression patterns of vimentin and glial filament protein with cytokeratins in the normal, hyperplastic, and neoplastic breast.

PubMed Central

Gould, V. E.; Koukoulis, G. K.; Jansson, D. S.; Nagle, R. B.; Franke, W. W.; Moll, R.

1990-01-01

The authors studied by immunohistochemistry the intermediate filament (IF) protein profile of 66 frozen samples of breast tissue, including normal parenchyma, all variants of fibrocystic disease (FCD), fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies (MAbs) to cytokeratins included MAb KA 1, which binds to polypeptide 5 in a complex with polypeptide 14 and recognizes preferentially myoepithelial cells; MAb KA4, which binds to polypeptides 14, 15, 16 and 19; individual MAbs to polypeptides 7, 13, and 16, 17, 18, and 19, and the MAb mixture AE1/AE3. The authors also applied three MAbs to vimentin (Vim), and three MAbs to glial filament protein (GFP). Selected samples were studied by double-label immunofluorescence microscopy and by staining sequential sections with some of the said MAbs, an MAb to alpha-smooth muscle actin, and well-characterized polyclonal antibodies for the possible coexpression of diverse types of cytoskeletal proteins. Gel electrophoresis and immunoblot analysis also were performed. All samples reacted for cytokeratins with MAbs AE1/AE3, although the reaction did not involve all cells. Monoclonal antibody KA4 stained preferentially the luminal-secretory cells in the normal breast and in FCD, whereas it stained the vast majority of cells in all carcinomas. Monoclonal antibody KA1 stained preferentially the basal-myoepithelial cells of the normal breast and FCD while staining tumor cell subpopulations in 4 of 31 carcinomas. Vimentin-positive cells were found in 8 of 12 normal breasts and in 12 of 20 FCD; in most cases, Vim-reactive cells appeared to be myoepithelial, but occasional luminal cells were also stained. Variable subpopulations of Vim-positive cells were noted in 9 of 20 ductal and in 1 of 7 lobular carcinomas. Glial filament protein-reactive cells were found in normal breast lobules and ducts and in 15 of 20 cases of FCD; with rare exceptions, GFP-reactivity was restricted to basally located, myoepithelial-appearing cells. Occasional GFP-reactive cells were found in 3 of 31 carcinomas. Evaluation of sequential sections and double-label immunofluorescence microscopy showed the coexpression of certain cytokeratins (possibly including polypeptides 14 and 17) with vimentin and alpha-smooth muscle actin together with GFP in some myoepithelial cells. The presence of GFP in myoepithelial cells was confirmed by gel electrophoresis and immunoblotting. Our results indicate that coexpression of cytokeratin with vimentin and/or GFP is comparatively frequent in normal basal-myoepithelial cells of the breast.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1700618

Spinal glucocorticoid receptor‑regulated chronic morphine tolerance may be through extracellular signal‑regulated kinase 1/2.

PubMed

Zhai, Mei-Li; Chen, Yi; Liu, Chong; Wang, Jian-Bo; Yu, Yong-Hao

2018-05-23

Opioid use has been limited in the treatment of chronic pain due to their side effects, including analgesic tolerance. Previous studies demonstrated that glucocorticoid receptors (GRs) may be involved in the development of chronic morphine tolerance; however, the mechanism remains unknown. It was hypothesized that the expression of spinal phosphorylated mitogen‑activated protein kinase [MAPK; phosphorylated extracellular signal‑regulated kinase (ERK)] is regulated through the spinal GRs, following chronic treatment with morphine. In the first experiment, the experimental rats were randomly divided into four groups: Control, morphine, morphine+GR antagonist mifepristone (RU38486) and morphine+GR agonist dexamethasone (Dex). Each group was treated with continuous intrathecal (IT) injection of the drugs for 6 days. The expression of GRs and MAPK 3/1 (p‑ERK 1/2) in the spinal dorsal horn was detected by western blot analysis and immunofluorescence staining. In the second experiment, the MAPK inhibitor PD98059 was added and the rats were randomly divided into four groups: Control, morphine, PD98059+morphine and PD98059+morphine+Dex. The continuous IT injection lasted for 7 days in each group. For all experiments, the tail flick test was conducted 30 min following administration every day to assess the thermal hyperalgesia of the rats. The experimental results demonstrated that there was a co‑existence of GRs and p‑ERK 1/2 in the spinal cord dorsal horn by double immunofluorescence staining. The GR antagonist RU38486 attenuated the morphine analgesia tolerance by inhibiting the expression of GR and increasing the expression of p‑ERK. The MAPK inhibitor PD98059 increased the effect of morphine tolerance and prolonged the duration of morphine tolerance. The present results suggest that spinal GRs may serve an important role in the development of morphine tolerance through the ERK signaling pathway.

Dynamic change of SGK expression and its role in neuron apoptosis after traumatic brain injury.

PubMed

Wu, Xinmin; Mao, Hui; Liu, Jiao; Xu, Jian; Cao, Jianhua; Gu, Xingxing; Cui, Gang

2013-01-01

Activation of specific signaling pathways in response to mechanical trauma causes delayed neuronal apoptosis; GSK-3β/β-catenin signaling plays a critical role in the apoptosis of neurons in CNS diseases, SGK was discovered as a regulator of GSK-3β/β-catenin pathway, The goal of this study was to determine if the mechanism of cell death or survival mediated by the SGK/GSK-3β/β-catenin pathway is involved in a rat model of TBI. Here, an acute traumatic brain injury model was applied to investigate the expression change and possible roles of SGK, Expression of SGK, and total-GSK-3β, phospho-GSK3β on ser-9, beta-catenin, and caspase-3 were examined by immunohistochemistry and Western blot analysis. Double immunofluorescent staining was used to observe the SGK localizations. Si-RNA was performed to identify whether SGK regulates neuron apoptosis via GSK-3β/β-catenin pathway, ultimately inhibit caspase-3 activation. Temporally, SGK expression showed an increase pattern after TBI and reached a peak at day 3. Spatially, SGK was widely expressed in the neuron, rarely in astrocytes and oligodendrocytes; in addition, the expression patterns of active caspase-3 and phospho-GSK3β were parallel with that of SGK, at the same time, the expression of β-catenin shows similarity with SGK. In vitro, to further investigate the function of SGK, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of SGK with apoptosis on PC12 cells by western blot, immunofluorescent labeling and siRNA. the results implied that SGK plays an important role in neuron apoptosis via the regulation of GSK3β/β-catenin signaling pathway; ultimately inhibit caspase-3 activation. Taken together, we inferred traumatic brain injury induced an upregulation of SGK in the central nervous system, which show a protective role in neuron apoptosis.

Forme particulière de Pemphigoide cicatricielle à dépôt unique d'IgA

PubMed Central

Aounallah, Amina; Jrad, Mariem; Ksiaa, Mehdi; Mokni, Sana; Saidi, Wafa; Boussofara, Lobna; Sriha, Badreddine; Denguezli, Mohamed; Ghariani, Najet; Belajouza, Colandane; Nouira, Rafia

2017-01-01

La Pemphigoide cicatricielle est une dermatose bulleuse sous épithéliale qui atteint essentiellement les muqueuses avec une évolution cicatricielle. Il s'agit d'un homme de 66 ans hospitalisé pour gingivite érosive avec dysphagie, dyspnée et flou visuel. L'examen dermatologique retrouvait des lésions érosives du palais et du pharynx. L'examen ophtalmologique notait des symblépharons, un ectropion et une cataracte bilatérale. La biopsie gingivale avait montré un décollement nécrotique de l'épithélium buccal. L'immunofluorescence directe notait un dépôt linéaire d'Immunoglobuline A à la jonction dermo-épidermique. L'Immunofluorescence indirecte était revenue négative. Le diagnostic de pemphigoide cicatricielle était confirmé. Le Transit oeso-gastro-duodénal a objectivé une double sténose de l'œsophage. L'endoscopie nasale, pharyngée et bronchique retrouvait des ulcérations de l'épiglotte, de l'hypopharynx, du pharynx et de l'arbre bronchique. Le patient a bénéficié d'un bolus de Solumedrol relayé par la Prednisone à la dose de 0.5mg/Kg/j associé à la Disulone à la dose de 100mg/j. L'évolution était favorable au début mais s'est compliquée après 2 mois d'une aggravation de la dysphagie et de la sténose oesophagienne. Notre observation est très particulière par la survenue d'une Pemphigoide cicatricielle chez un sujet de sexe masculin ayant un tableau grave en rapport avec l'extension des lésions à toutes les muqueuses conjonctivale, buccale, nasale, œsophagienne et même bronchique associée à une immunofluorescence directe faite d'un dépôt d'IgA uniquement. PMID:28533859

Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus.

PubMed

Launay, David; Schmidt, Jean; Lepers, Sébastien; Mirault, Tristan; Lambert, Marc; Kyndt, Xavier; Reumaux, Dominique; Duhamel, Alain; Hachulla, Eric; Hatron, Pierre-Yves; Prin, Lionel; Dubucquoi, Sylvain

2010-07-04

Among anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE). Anti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases. Farr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE. Farr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment. Copyright 2010 Elsevier B.V. All rights reserved.

[Substance P and/or calcitonin gene-related peptide immunoreactive neurons in dorsal root ganglia possibly involved in the transmission of nociception in rat penile frenulum].

PubMed

Wu, Zhong-Min; Ni, Jing-Jing; Ling, Shu-Cai

2007-12-01

To study the relationship between substance P (SP) and/or calcitonin gene-related peptide (CGRP) immunoreactive neurons in dorsal root ganglia (DRG) and the transmission of nociception in the penile frenulum of rats. The fluoro-gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in the penile frenulum of rats. And SP and/or CGRP immunofluorescence labeling was employed to detect the distribution of SP and/or CGRP immunoreactive neurons in DRG. FG retrograde tracing showed that the FG retrolabeled neurons were localized in L6-DRG and S1-DRG. SP and/or CGRP immunofluorescence labeling indicated that a large number of DRG neurons were SP- and CGRP-immunoreactive, different in size, bright red and bright green respectively in color, and arranged in rows or spots among nerve bundles. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L6-DRG and S1-DRG respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro labeled neurons. SP- and CGRP-immunoreactive neurons in L6-DRG and SI-DRG of rats may be involved in the transmission of nociception in rat penile frenulum.

[Prokaryotic expression and histological localization of the Taenia solium CDC37 gene].

PubMed

Huang, Jiang; Li, Bo; Dai, Jia-Lin; Zhang, Ai-Hua

2013-02-01

To express Taenia solium gene encoding cell division cycle 37 protein (TsCDC37) and investigate its antigenicity and localization in adults of Taenia solium. The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a (+). The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund's adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique. The recombinant expression vector was constructed successfully. The recombinant protein was about M(r) 52 000, it was then purified and specifically recognized by immuno sera of SD rats and sera from patients infected with Taenia solium, Taenia saginata or Taenia asiatica. The immunofluorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs. TsCDC37 gene has been expressed with immunoreactivity. The recombinant protein is mainly expressed in tegument and egg, and is a common antigen of the three human taenia cestodes.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

PubMed

Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Ibaragi, Soichiro; Kunisada, Yuki; Obata, Kyoichi; Masui, Masanori; Pai, Pang; Horikiri, Yuu; Yamanaka, Nobuyuki; Takigawa, Masaharu; Sasaki, Akira

2018-06-01

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling. © 2017 Wiley Periodicals, Inc.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

PubMed Central

Gojanovich, Aldana D.; Gimenez, María C.; Masone, Diego; Rodriguez, Tania M.; Dewey, Ricardo A.; Delgui, Laura R.; Bustos, Diego M.; Uhart, Marina

2018-01-01

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications. PMID:29670879

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining.

PubMed

Gojanovich, Aldana D; Gimenez, María C; Masone, Diego; Rodriguez, Tania M; Dewey, Ricardo A; Delgui, Laura R; Bustos, Diego M; Uhart, Marina

2018-01-01

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

The relationship of fibroblast translocations to cell morphology and stress fibre density.

PubMed

Lewis, L; Verna, J M; Levinstone, D; Sher, S; Marek, L; Bell, E

1982-02-01

Translocation of human fibroblasts in culture was studied using techniques of time-lapse cinemicrography, indirect immunofluorescence, and computer analysis. An inverse relationship between the velocity of cells during the last hour of life and the density of stress fibers seen by immune staining was demonstrated. Translocating cells generally assumed one of two interconvertible morphologies: a triangular tailed shape or tailed fibroblast (TF), and a tailless form that resembled a half-moon, which we call a half-moon fibroblast (HMF). The tail of TFs formed only on regions of substrate that had been previously traversed by cells. The half-moon morphology developed either on previously used or on virgin substrate. Cells adopted the HMF rather than the TF morphology with a four-fold greater frequency. HMFs translocated slightly faster than TFs. The foregoing observation suggest that the fibroblast tail is not an organelle essential for translocation. Since our technique allowed us to distinguish between cells which were cycling and those which had left cycle, we compared their velocities and found them to be similar. Also the average velocities of cells of different population-doubling levels (10th, 30th, 40th) were approximately equal.

Increased expression of EMMPRIN and VEGF in the rat brain after gamma irradiation.

PubMed

Wei, Ming; Li, Hong; Huang, Huiling; Xu, Desheng; Zhi, Dashi; Liu, Dong; Zhang, Yipei

2012-03-01

The extracellular matrix metalloproteinase inducer (EMMPRIN) has been known to play a key regulatory role in pathological angiogenesis. A elevated activation of vascular endothelial growth factor (VEGF) following radiation injury has been shown to mediate blood-brain barrier (BBB) breakdown. However, the roles of EMMPRIN and VEGF in radiation-induced brain injury after gamma knife surgery (GKS) are not clearly understood. In this study, we investigated EMMPRIN changes in a rat model of radiation injury following GKS and examined potential associations between EMMPRIN and VEGF expression. Adult male rats were subjected to cerebral radiation injury by GKS under anesthesia. We found that EMMPRIN and VEGF expression were markedly upregulated in the target area at 8-12 weeks after GKS compared with the control group by western blot, immunohistochemistry, and RT-PCR analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals colocalized with caspase-3 and VEGF-positive cells. Our data also demonstrated that increased EMMPRIN expression was correlated with increased VEGF levels in a temporal manner. This is the first study to show that EMMPRIN and VEGF may play a role in radiation injuries of the central nervous system after GKS.

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

PubMed Central

Bezek, D M; Baker, J C; Kaneene, J B

1988-01-01

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen. PMID:2836047

Forkhead box-P3+ regulatory T cells and toll-like receptor 2 co-expression in oral squamous cell carcinoma.

PubMed

Hussaini, H M; Parachuru, V P B; Seymour, G J; Rich, A M

2017-04-01

The function of forkhead box-P3 (FoxP3) regulatory T cells (Treg) and toll-like receptor (TLR)2 protein in the oral cancer microenvironment is not fully understood, but evidence from other malignancies suggests it is likely they are involved with tumour development and progression. The aim of this study was to investigate the distribution of FoxP3 + cells, TLR2 + cells and double-labelled FoxP3 + TLR2 + immune cells in oral squamous cell carcinoma (OSCC), using immunohistochemistry (IHC) and immunofluorescence (IF). 25 archival cases of OSCC were immunostained with anti-FoxP3 and anti-TLR2 antibodies. Inflamed hyperplastic oral mucosal tissues were used as controls. The proportion of single-labelled, double-labelled and negative cells was determined. A higher frequency of double-labelled FoxP3 + TLR2 + Tregs was observed within the immune cells of OSCC compared to inflamed controls using IHC (p<0.05). Cell-to-cell contact between single-stained TLR2 + cells and FoxP3 + cells was noted. Double IF studies validated demonstration of co-expression of FoxP3 + /TLR2 + immune cells in OSCC. The presence of FoxP3 + TLR2 + cells within the OSCC microenvironment may represent a dendritic cell-dependent pathway capable of inhibiting Treg suppressive activity, potentially enhancing the anti-tumour response. Modulation of TLR2-Treg interactions should be further explored to determine if they have a role in the therapeutic management of OSCC. Copyright © 2017 Elsevier GmbH. All rights reserved.

Sudan Black B masks Mycobacterium avium subspecies paratuberculosis immunofluorescent antibody labeling

USDA-ARS?s Scientific Manuscript database

Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

[Effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier of rats with acute carbon monoxide poisoning].

PubMed

Wang, Li; Ding, Xiaoyu; Bi, Mingjun; Wang, Jinglin; Zou, Yong; Tang, Jiyou; Li, Qin

2018-05-01

To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning. A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression. The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein: 3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein: 20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R 2 = 0.917, P = 0.022). NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.

PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

PubMed

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410-fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

Th17-cells in atopic dermatitis stimulate orthodontic root resorption.

PubMed

Yamada, K; Yamaguchi, M; Asano, M; Fujita, S; Kobayashi, R; Kasai, K

2013-10-01

The aim of this study was to investigate how atopic dermatitis (AD) contributes to root resorption during orthodontic tooth movement. Atopic dermatitis model mice and wild-type mice were subjected to an excessive orthodontic force (OF) to induce movement of the upper first molars. The expression levels of the tartrate-resistant acid phosphatase (TRAP), IL-17, IL-6, and RANKL proteins were determined in the periodontal ligament (PDL) by an immunohistochemical analysis. Furthermore, the effects of the compression force on co-cultures of CD4(+) cells from AD patients or healthy individuals and human PDL cells were investigated with regard to the levels of secretion and mRNA expression of IL-17, IL-6, RANKL, and osteoprotegerin. The immunoreactivities for TRAP, IL-17, IL-6, and RANKL in the AD group were found to be significantly increased. The double immunofluorescence analysis for IL-17/CD4 detected immunoreaction. The secretion of IL-17, IL-6, and RANKL, and the mRNA levels of IL-6 and RANKL in the AD patients were increased compared with those in healthy individuals. Th17 cells may therefore be associated with the deterioration of root resorption of AD mice, and may explain why AD patients are more susceptible to root resorption than healthy individuals when an excessive OF is applied. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

An automated approach to the segmentation of HEp-2 cells for the indirect immunofluorescence ANA test.

PubMed

Tonti, Simone; Di Cataldo, Santa; Bottino, Andrea; Ficarra, Elisa

2015-03-01

The automatization of the analysis of Indirect Immunofluorescence (IIF) images is of paramount importance for the diagnosis of autoimmune diseases. This paper proposes a solution to one of the most challenging steps of this process, the segmentation of HEp-2 cells, through an adaptive marker-controlled watershed approach. Our algorithm automatically conforms the marker selection pipeline to the peculiar characteristics of the input image, hence it is able to cope with different fluorescent intensities and staining patterns without any a priori knowledge. Furthermore, it shows a reduced sensitivity to over-segmentation errors and uneven illumination, that are typical issues of IIF imaging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tomato Spotted Wilt Virus Particle Morphogenesis in Plant Cells

PubMed Central

Kikkert, Marjolein; Van Lent, Jan; Storms, Marc; Bodegom, Pentcho; Kormelink, Richard; Goldbach, Rob

1999-01-01

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed. PMID:9971812

Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.

PubMed

Bloemberg, Darin; Quadrilatero, Joe

2012-01-01

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles.

Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

PubMed Central

Wyroba, E.; Kwaśniak, P.; Miller, K.; Kobyłecki, K.; Osińska, M.

2016-01-01

Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P32] by 37% and of [C14-]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.63+ representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P32] and [C14]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PMID:27349314

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells.

PubMed

Le, Wei; Qi, Lixin; Li, Jiaxuan; Wu, DengIong; Xu, Jun; Zhang, Jinfu

2016-01-01

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Carbon Limitation Induces ςS-Dependent Gene Expression in Pseudomonas fluorescens in Soil

PubMed Central

Koch, Birgit; Worm, Jakob; Jensen, Linda E.; Højberg, Ole; Nybroe, Ole

2001-01-01

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the ςS-dependent Escherichia coli promoter Pfic and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of β-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing β-galactosidase from Pfic. Changes in cell size and expression of β-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of β-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil β-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted. PMID:11472905

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

PubMed Central

Gerritsen, L.; van der Wolf, J. M.; van Vuurde, J.; Ehlers, R.; Krasomil-Osterfel..., K. C.; Smits, P. H.

1995-01-01

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture. PMID:16534911

IFDOTMETER: A New Software Application for Automated Immunofluorescence Analysis.

PubMed

Rodríguez-Arribas, Mario; Pizarro-Estrella, Elisa; Gómez-Sánchez, Rubén; Yakhine-Diop, S M S; Gragera-Hidalgo, Antonio; Cristo, Alejandro; Bravo-San Pedro, Jose M; González-Polo, Rosa A; Fuentes, José M

2016-04-01

Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier. © 2015 Society for Laboratory Automation and Screening.

Pure laparoscopic hepatectomy with augmented reality-assisted indocyanine green fluorescence versus open hepatectomy for hepatocellular carcinoma with liver cirrhosis: A propensity analysis at a single center.

PubMed

Cheung, Tan To; Ma, Ka Wing; She, Wong Hoi; Dai, Wing Chiu; Tsang, Simon Hing Yin; Chan, Albert Chi Yan; Chok, Kenneth Siu Ho; Lo, Chung Mau

2018-05-10

Laparoscopic hepatectomy is considered an acceptable treatment of choice in selected patients with primary hepatocellular carcinoma (HCC). Whether indocyanine green (ICG) immunofluorescence, a new technology, may improve surgery outcomes has yet to be tested. The aim of the present study was to investigate and compare the effect of ICG fluorescence imaging on the outcomes of pure laparoscopic hepatectomy and open hepatectomy for primary HCC with background cirrhosis. From January 2015 to June 2016, 20 patients with HCC and liver cirrhosis underwent laparoscopic hepatectomy with ICG immunofluorescence. The outcomes of pure laparoscopic hepatectomy with ICG immunofluorescence were compared with those of open hepatectomy. To avoid selection bias, patients were propensity score matched in a ratio of 1 : 6, with 20 patients in the laparoscopic group and 120 in the open group. The laparoscopic group had 20 patients, and the open group had 120 patients. The laparoscopic group had less blood loss (125 vs 450 mL, P < 0.001), a shorter operation time (200 vs 250 min, P = 0.003), and a shorter hospital stay (5 vs 6 days, P < 0.001). The complication rate was 0% in the laparoscopic group compared to 15.0% in the open group (P = 0.135). All patients in the laparoscopic group had negative margin involvement. Four patients (3.3%) in the open resection group had positive margin involvement. Two patients in the ICG immunofluorescence group had additional lesions identified and resected during operation. Pure laparoscopic hepatectomy with ICG immunofluorescence for primary HCC can be carried out safely with favorable short-term outcomes even in cirrhotic patients. Better identification of the bile duct structure and better assessment of the tumor resection margin and perfusion are advantages of this new technique. © 2018 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and John Wiley & Sons Australia, Ltd.

Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

PubMed Central

Frost, Emily; Dixon, Mellisa; Ollosson, Sarah; Kilpin, Kate; Patel, Mitali; Scully, Juliet; Rogers, Andrew V.; Mitchison, Hannah M.; Bush, Andrew; Hogg, Claire

2017-01-01

Rationale: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom consists of assessing ciliary function by high-speed microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of biallelic disease-causing mutations is also diagnostic, but many disease-causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. Objectives: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. Methods: The study used immunofluorescent labeling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared with diagnostic outcome. Measurements and Main Results: Immunofluorescence correctly identified mislocalized or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared with electron microscopy (27 days; cost $1,452). Conclusions: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test. PMID:28199173

rhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells.

PubMed

Wang, Huqing; Fan, Jiaxin; Chen, Mengyi; Yao, Qingling; Gao, Zhen; Zhang, Guilian; Wu, Haiqin; Yu, Xiaorui

2017-08-01

Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100 U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti-oxidant capacity. These findings provide a theoretical basis to further the anti-aging mechanism of EPO in the nervous system, and they provide experimental evidence at the cellular level for the clinical application of EPO to protect the nervous system from aging.

Changes of immunocytochemical localization of vesicular glutamate transporters in the rat visual system after the retinofugal denervation.

PubMed

Fujiyama, Fumino; Hioki, Hiroyuki; Tomioka, Ryohei; Taki, Kousuke; Tamamaki, Nobuaki; Nomura, Sakashi; Okamoto, Keiko; Kaneko, Takeshi

2003-10-13

To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1- and VGluT2-immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1-immunolabeled terminals were smaller than VGluT2-labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra- and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons. Copyright 2003 Wiley-Liss, Inc.

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.

PubMed

MacKenzie, Tippi C; Kobinger, Gary P; Louboutin, Jean-Pierre; Radu, Antoneta; Javazon, Elizabeth H; Sena-Esteves, Miguel; Wilson, James M; Flake, Alan W

2005-01-01

We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

The adaptor protein p62 is involved in RANKL-induced autophagy and osteoclastogenesis.

PubMed

Li, Rui-Fang; Chen, Gang; Ren, Jian-Gang; Zhang, Wei; Wu, Zhong-Xing; Liu, Bing; Zhao, Yi; Zhao, Yi-Fang

2014-12-01

Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases. © The Author(s) 2014.

Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells.

PubMed

Sorkio, Anni E; Vuorimaa-Laukkanen, Elina P; Hakola, Hanna M; Liang, Huamin; Ujula, Tiina A; Valle-Delgado, Juan José; Österberg, Monika; Yliperttula, Marjo L; Skottman, Heli

2015-05-01

The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruch's membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biological characterization of metanephric mesenchymal stem cells from the Beijing duck.

PubMed

Chen, Jia; Pu, Yabin; Sun, Yujiao; Zhang, Ping; Li, Qian; Wang, Kunfu; Wang, Wenjie; Ma, Yuehui; Guan, Weijun

2016-02-01

Mesenchymal stem cells (MSCs) possess self-proliferation and multi-directional differentiation abilities. Previous studies on MSCs have mostly focused on the bone marrow, lungs, pancreas and umbilical cord blood, with few studies on metanephric tissues in ducks. For the present study, the Beijing duck was selected as an experimental animal. Duck embryo metanephric mesenchymal stem cells (MMSCs) were studied. MMSC isolation culture, analysis of biological characteristics, induced differentiation and identification were performed in preliminary experiments. In the current study, surface antigens and gene expression patterns were detected using immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. The induced cells, adipocytes, hepatocytes, epithelial cells and islet cells were identified by oil red O staining, periodic acid-Schiff staining, immunofluorescence and dithizone staining, respectively. RT-PCR was performed for detection of specific marker genes. The results suggested that the biological characteristics of MMSCs were similar to those of the MSCs previously analyzed. Primary MMSCs were sub-cultured to passage 21. The induced cells exhibit typical staining and immunofluorescence indicating the expression of specific genes. This demonstrates that MMSCs may be a novel alternative source of MSCs for experimental and clinical applications.

Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

NASA Astrophysics Data System (ADS)

Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

2018-02-01

Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

NASA Technical Reports Server (NTRS)

Chakrabarti, Amitabha; Stoecker, Andrew; Schatten, Heide

1995-01-01

To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

PubMed Central

Lv, Yun-Cheng; Wang, Zong-Bao; Yao, Feng; Xie, Wei; Tan, Yu-Lin; Li, Liang; Zhang, Min; Lan, Gang; Gong, Duo; Cheng, Hai-Peng; Zhong, Hui-Juan; Liu, Dan; Huang, Chong; Li, Zhao-Xia; Zheng, Xi-Long; Yin, Wei-Dong; Tang, Chao-Ke

2015-01-01

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion. PMID:26397958

Central nervous system damage due to acute paraquat poisoning: an experimental study with rat model.

PubMed

Wu, Bailin; Song, Bo; Yang, Haiqing; Huang, Boyuan; Chi, Bo; Guo, Yansu; Liu, Huaijun

2013-03-01

Paraquat (PQ) is a common herbicide and PQ poisoning is a major medical problem in Asia. However, few studies have focused on the acute neurotoxic changes caused by PQ. Here we report the acute neurotoxicological findings of rats treated with lethal dose of PQ. In substantia nigra (SN) and striatum we found obvious microglia (labeled by Iba-1) activation within one week. In SN and hippocampus, we detected increased oxidative stress in the neurons based on NeuN/8-OHdG immunofluorescence double labeling and laser cofocal microscopy. Moreover, we provided ultrastructural evidences of astrocyte edema and neurons apoptosis in rat brain by electron microscopy. Further studies will be needed with non-lethal dose of PQ to confirm these results and demonstrate the direct CNS toxicity of PQ. Copyright © 2012 Elsevier Inc. All rights reserved.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis.

PubMed

Del Negro, G M; Garcia, N M; Rodrigues, E G; Cano, M I; de Aguiar, M S; Lírio, V de S; Lacaz, C da S

1991-01-01

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e., a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO. The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

PubMed

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Neurovascular and Neuroimmune Aspects in the Pathophysiology of Rosacea

PubMed Central

Schwab, Verena D.; Sulk, Mathias; Seeliger, Stephan; Nowak, Pawel; Aubert, Jerome; Mess, Christian; Rivier, Michel; Carlavan, Isabelle; Rossio, Patricia; Metze, Dieter; Buddenkotte, Jörg; Cevikbas, Ferda; Voegel, Johannes J.; Steinhoff, Martin

2013-01-01

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea. PMID:22076328

Absence of convincing evidence of Coxiella burnetii infection in Chile: a cross-sectional serosurvey among healthy adults in four different regions.

PubMed

Weitzel, Thomas; López, Javier; Acosta-Jamett, Gerardo; Edouard, Sophie; Parola, Philippe; Abarca, Katia

2016-10-06

Coxiella burnetii is an important zoonotic pathogen of global distribution. Still, in most parts of South America including Chile, systematic epidemiological data are lacking. The presented study aims to determine the seroprevalence of Coxiella burnetii antibodies in healthy adults of four different regions in Chile. A cross-sectional study was performed, which included healthy adults living in rural and urban areas of four cities located in different regions in northern, central, and southern Chile. In urban sectors, households were chosen by double stratified random sampling, while in rural areas convenience sampling was performed. Serum specimens were taken and screened for the presence of IgG antibodies against C. burnetii phase II antigen using a commercial ELISA kit. Positive and indeterminate results were confirmed by a reference laboratory using indirect immunofluorescence assay (IFA). A total of 1112 individuals were included. Of those, 8 were positive by ELISA, but only one sample was confirmed using IFA. Statistical analysis for population freedom from disease revealed a high probability that C. burnetii was absent in our study population. Our work provides the first epidemiological data on human Q fever in Chile indicating either a very low endemicity or the absence of this pathogen in the studied areas.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

OUR EXPERIENCE WITH IMMUNOFLUORESCENCE (RELATING TO 250 SERA STUDIED AT THE NANCY DERMATOLOGICAL CLINIC)

DTIC Science & Technology

The principle of immunofluorescence, discovered by Coons in 1942, was applied to the diagnosis of syphilis by Deacon, Falcon and Harris in 1957...technique and to determine its value (Dauget, Fribourg-Blanc, Thivolet). The research reported here, relating to 250 sera, seems to confirm the great importance of the immunofluorescence test (IF) in the diagnosis of syphilis .

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

The roles of RUNX3 in cervical cancer cells in vitro.

PubMed

Li, Zhen; Fan, Pan; Deng, Min; Zeng, Chao

2018-06-01

RUNX3 serves an important role in development of various types of human cancer. The purpose of the present study was to investigate the potential biological function of RUNX3 in cervical cancer cells. In the present study, a RUNX3 overexpressed model was constructed in Hec1 cells by PCDNA3.1-RUNX3 transfection. Western blot analysis was used to measure RUNX3 expression in cervical cancer cells. Immunofluorescence analysis was performed to examine subcellular localization of RUNX3 in cervical cancer cells. Effects of RUNX3 expression on proliferation, migration and invasion of cervical cancer cells were detected by colony formation assay, wound healing assay and Transwell assay, respectively. Immunofluorescence confirmed the nuclear location of RUNX3 in cervical cancer cell. Result sindicated that upregulation of RUNX3 expression inhibited proliferation, migration and invasion of cervical cancer cells. However, knockdown of RUNX3 expression promoted the proliferation, migration and invasion of cervical cancer cells. Hence, RUNX3 may serve as a tumor suppressor gene in cervical cancer.

The Analysis of the Patterns of Radiation-Induced DNA Damage Foci by a Stochastic Monte Carlo Model of DNA Double Strand Breaks Induction by Heavy Ions and Image Segmentation Software

NASA Technical Reports Server (NTRS)

Ponomarev, Artem; Cucinotta, F.

2011-01-01

To create a generalized mechanistic model of DNA damage in human cells that will generate analytical and image data corresponding to experimentally observed DNA damage foci and will help to improve the experimental foci yields by simulating spatial foci patterns and resolving problems with quantitative image analysis. Material and Methods: The analysis of patterns of RIFs (radiation-induced foci) produced by low- and high-LET (linear energy transfer) radiation was conducted by using a Monte Carlo model that combines the heavy ion track structure with characteristics of the human genome on the level of chromosomes. The foci patterns were also simulated in the maximum projection plane for flat nuclei. Some data analysis was done with the help of image segmentation software that identifies individual classes of RIFs and colocolized RIFs, which is of importance to some experimental assays that assign DNA damage a dual phosphorescent signal. Results: The model predicts the spatial and genomic distributions of DNA DSBs (double strand breaks) and associated RIFs in a human cell nucleus for a particular dose of either low- or high-LET radiation. We used the model to do analyses for different irradiation scenarios. In the beam-parallel-to-the-disk-of-a-flattened-nucleus scenario we found that the foci appeared to be merged due to their high density, while, in the perpendicular-beam scenario, the foci appeared as one bright spot per hit. The statistics and spatial distribution of regions of densely arranged foci, termed DNA foci chains, were predicted numerically using this model. Another analysis was done to evaluate the number of ion hits per nucleus, which were visible from streaks of closely located foci. In another analysis, our image segmentaiton software determined foci yields directly from images with single-class or colocolized foci. Conclusions: We showed that DSB clustering needs to be taken into account to determine the true DNA damage foci yield, which helps to determine the DSB yield. Using the model analysis, a researcher can refine the DSB yield per nucleus per particle. We showed that purely geometric artifacts, present in the experimental images, can be analytically resolved with the model, and that the quantization of track hits and DSB yields can be provided to the experimentalists who use enumeration of radiation-induced foci in immunofluorescence experiments using proteins that detect DNA damage. An automated image segmentaiton software can prove useful in a faster and more precise object counting for colocolized foci images.

PmVRP15, a Novel Viral Responsive Protein from the Black Tiger Shrimp, Penaeus monodon, Promoted White Spot Syndrome Virus Replication

PubMed Central

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway. PMID:24637711

Establishment and characterization of a new human acinar cell carcinoma cell line, Faraz-ICR, from pancreas.

PubMed

Rezaei, Marzieh; Hosseini, Ahmad; Nikeghbalian, Saman; Ghaderi, Abbas

Basic research in the field of acinar cell carcinoma (ACC) as a rare neoplasm of the pancreas is dependent on the availability of pragmatic model such as new pancreatic cancer cell lines. Thus, establishment and characterization of new pancreatic cancer cell lines from ACC origin are deemed important. Faraz-ICR cell line was derived from a 58-years old woman with pancreatic acinar cell carcinoma by the collagenase digestion protocol. We characterized the cell line by examining its morphology and cytostructural and functional profile. Faraz-ICR has a doubling time of 35 hours and grows in soft agar with a colony-forming efficiency of 25%. The cell had nearly normal pattern of chromosomes in karyotype analysis and Comparative Genomic Hybridization (CGH) array analysis. Evaluation of cells by flowcytometry showed that Faraz-ICR is negative for EpCAM and mesenchymal markers in different passages, and has epithelial nature. Immunofluorescence staining revealed that cells were strongly positive for vimentin, desmin, ezrin, S100, nestin and they were negative for pan-cytokeratins, chromogranin and alpha smooth muscle actin. We were able to establish a new pancreatic carcinoma cell line with partial aspects of Epithelial-mesenchymal transition and aggressiveness. This cell line might be suitable for studying various anticancer drugs and protein profile aiming to see any possible tumor associated marker for ACC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4.

PubMed

Salewsky, Bastian; Schmiester, Maren; Schindler, Detlev; Digweed, Martin; Demuth, Ilja

2012-11-15

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo.

PubMed

Moyen, Nanikaly; Thirion, Laurence; Emmerich, Petra; Dzia-Lepfoundzou, Amelia; Richet, Hervé; Boehmann, Yannik; Dimi, Yannick; Gallian, Pierre; Gould, Ernest A; Günther, Stephan; de Lamballerie, Xavier

2015-01-01

Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV. In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds. This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.

Involvement of microRNA-181a and Bim in a rat model of retinal ischemia-reperfusion injury.

PubMed

He, Yu; Liu, Jin-Nan; Zhang, Jun-Jun; Fan, Wei

2016-01-01

To investigate the changes in the expression of microRNA-181a (miR-181a) and Bim in a rat model of retinal ischemia-reperfusion (RIR), to explore their target relationship in RIR and their involvement in regulating apoptosis of retinal ganglion cells (RGCs). Target gene prediction for miR-181a was performed with the aid of bioinformatics and Bim was identified as a potential target gene of miR-181a. A rat model of RIR was created by increasing the intraocular pressure. RGCs in the flatmounted retinas were labeled with Brn3, a marker for alive RGCs, by immunofluorescent staining. The changes in the number of RGCs after RIR were recorded. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression level of miR-181a in the retina. Bim/Brn3 double immunofluorescence was used to detect the localization of Bim. The expression of Bim in the retina was determined with the aids of Western blot and qRT-PCR. Compared with the negative control group, the density of RGCs was significantly lower in the ischemia/reperfusion (I/R)-24h and I/R-72h groups (P<0.001). The expression level of miR-181a started to decrease at 0h after RIR, and further decreased at 24h and 72h compared with the negative control group (P<0.001). Bim was significantly upregulated at 12h after RIR (P<0.05) and reached peak at 24, 72h compared with the negative control group (P<0.01). Pearson correlation analysis showed that the expression level of Bim was negatively correlated with the expression level of miR-181a and the density of RGCs. Bim may be a potential target gene of miR-181a. Both miR-181a and Bim are involved in RGCs death in RIR. RIR may promote RGCs apoptosis in the retina via downregulation of miR-181a and its inhibition on Bim expression.

Soluble FGL2, a novel effector molecule of activated hepatic stellate cells, regulates T-cell function in cirrhotic patients with hepatocellular carcinoma.

PubMed

Sun, Ying; Xi, Dong; Ding, Wen; Wang, Faxi; Zhou, Haili; Ning, Qin

2014-10-01

To investigate the effects of soluble FGL2 (sFGL2) secreted by hepatic stellate cells (HSCs) on immune suppression in cirrhotic patients with hepatocellular carcinoma (HCC). Serum sFGL2 levels were examined by ELISA in 40 patients with HCC, liver cirrhosis (LC) or chronic HBV (CHB) infection. A double staining of the immunofluorescence analysis of α-SMA and FGL2 was performed in two cirrhotic liver specimens. The expression of FGL2 in the LX2 cell line was analyzed by immunofluorescence, Western blot and flow cytometry. T-cells purified from HCC patients using magnetic beads were cultured with LX2 cells at different ratios with anti-CD3-stimulating or FGL2-blocking antibodies. The proliferation index (PI) of CD8 + T cells was assessed by flow cytometry, and the secretion of IFN-γ was measured by ELISA. sFGL2 levels are significantly higher in patients with HCC or LC compared with those with CHB (p = 0.0039/p = 0.0020). Among HCC patients, those with cirrhosis exhibited significantly higher levels of sFGL2 compared with non-cirrhotic individuals (p = 0.0108). The expressions of FGL2 and α-SMA overlapped in HSCs in liver specimens. FGL2 protein secreted by LX2 cells inhibited T-cell proliferation of HCC patients in a dose-dependent manner in vitro. The PI of CD8 + T cells was significantly enhanced following addition of FGL2 antibody to the culture system (LX2/T-cell ratio of 1:10, p = 0.002). The level of IFN-γ in mixed cultures was inversely correlated with the number of HSCs and was reversed by incubation with FGL2 blocking antibody. sFGL2 protein is a novel effector molecule of activated HSCs, which suppresses CD8 + T cell proliferation and interferon-γ production, and it subsequently might contribute to immune suppression during fibrosis and tumorigenesis in the liver.

Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

PubMed

Boggild, A K; Sundermann, C A; Estridge, B H

2002-01-01

Glutamylated and tyrosinated tubulin were localized in Giardia intestinalis and selected trichomonads of the Tritrichomonadinae subfamily, using specific monoclonal antibodies directed at each of the post-translational modifications. Analysis was carried out using indirect immunofluorescence microscopy. Although trichomonad tubulins remained unlabeled by anti-tyrosine tubulin (TUB-1A2), the presence of the glutamylation motif (GT 335) was confirmed and found to differ in distribution among tritrichomonads. Tritrichomonas muris was most heavily labeled with GT 335, while T. foetus was the least so. Like trichomonads, Giardia was unreactive to anti-tyrosine tubulin; however, the GT 335 antibody produced marked fluorescence in Giardia trophozoites. This study is the first to report immunofluorescent localization of tubulin glutamylation in Giardia and confirms previously reported mass spectrometry data.

Flow cytometric immunofluorescence of rat anterior pituitary cells

NASA Technical Reports Server (NTRS)

Hatfield, J. Michael; Hymer, W. C.

1985-01-01

A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine.

PubMed Central

Armstrong, C H; Scheidt, A B; Thacker, H L; Runnels, L J; Freeman, M J

1984-01-01

Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally. PMID:6434167

Improving DNA double-strand repair inhibitor KU55933 therapeutic index in cancer radiotherapy using nanoparticle drug delivery.

PubMed

Tian, Xi; Lara, Haydee; Wagner, Kyle T; Saripalli, Srinivas; Hyder, Syed Nabeel; Foote, Michael; Sethi, Manish; Wang, Edina; Caster, Joseph M; Zhang, Longzhen; Wang, Andrew Z

2015-12-21

Radiotherapy is a key component of cancer treatment. Because of its importance, there has been high interest in developing agents and strategies to further improve the therapeutic index of radiotherapy. DNA double-strand repair inhibitors (DSBRIs) are among the most promising agents to improve radiotherapy. However, their clinical translation has been limited by their potential toxicity to normal tissue. Recent advances in nanomedicine offer an opportunity to overcome this limitation. In this study, we aim to demonstrate the proof of principle by developing and evaluating nanoparticle (NP) formulations of KU55933, a DSBRI. We engineered a NP formulation of KU55933 using nanoprecipitation method with different lipid polymer nanoparticle formulation. NP KU55933 using PLGA formulation has the best loading efficacy as well as prolonged drug release profile. We demonstrated that NP KU55933 is a potent radiosensitizer in vitro using clonogenic assay and is more effective as a radiosensitizer than free KU55933 in vivo using mouse xenograft models of non-small cell lung cancer (NSCLC). Western blots and immunofluorescence showed NP KU55933 exhibited more prolonged inhibition of DNA repair pathway. In addition, NP KU55933 leads to lower skin toxicity than KU55933. Our study supports further investigations using NP to deliver DSBRIs to improve cancer radiotherapy treatment.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

PubMed

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-05-10

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats

PubMed Central

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-01-01

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. PMID:28489060

Grape seed extract enhances neurogenesis in the hippocampal dentate gyrus in C57BL/6 mice.

PubMed

Yoo, Dae Young; Kim, Woosuk; Yoo, Ki-Yeon; Lee, Choong Hyun; Choi, Jung Hoon; Yoon, Yeo Sung; Kim, Dong-Woo; Won, Moo-Ho; Hwang, In Koo

2011-05-01

The effects of grape seed extract (GSE), a major source of phenolic compounds, were examined on cell proliferation, neuroblast differentiation and integration into granule cells in the hippocampal dentate gyrus (DG) of middle-aged (12 month-old) mice using Ki67, doublecortin (DCX) immunohistochemistry and 5'-bromo-2-deoxyguanosine (BrdU)/calbindin D-28k (CB) double immunofluorescence study, respectively. GSE (25, 50 and 100 mg/kg) was administered orally for 28 days, and the animals were treated with 50 mg/kg BrdU intraperitoneally on the day of first GSE treatment. In the vehicle-treated group, Ki67 and DCX immunoreactivity was detected in the subgranular zone of the DG (SZDG). GSE treatment dose-dependently increased the number of Ki67 and DCX immunoreactive cells, particularly the number of DCX immunoreactive neuroblasts with well-developed (tertiary) dendrites. GSE also dose-dependently increased DCX protein levels. In addition, GSE treatment increased significantly the number of BrdU/CB double labeled granule cells. These results suggest that GSE significantly increases cell proliferation, neuroblast differentiation and integration into granule cells in the DG, and the consumption of GSE enhances the plasticity of hippocampus in middle-aged mice. Copyright © 2010 John Wiley & Sons, Ltd.

Improving DNA double-strand repair inhibitor KU55933 therapeutic index in cancer radiotherapy using nanoparticle drug delivery

NASA Astrophysics Data System (ADS)

Tian, Xi; Lara, Haydee; Wagner, Kyle T.; Saripalli, Srinivas; Hyder, Syed Nabeel; Foote, Michael; Sethi, Manish; Wang, Edina; Caster, Joseph M.; Zhang, Longzhen; Wang, Andrew Z.

2015-11-01

Radiotherapy is a key component of cancer treatment. Because of its importance, there has been high interest in developing agents and strategies to further improve the therapeutic index of radiotherapy. DNA double-strand repair inhibitors (DSBRIs) are among the most promising agents to improve radiotherapy. However, their clinical translation has been limited by their potential toxicity to normal tissue. Recent advances in nanomedicine offer an opportunity to overcome this limitation. In this study, we aim to demonstrate the proof of principle by developing and evaluating nanoparticle (NP) formulations of KU55933, a DSBRI. We engineered a NP formulation of KU55933 using nanoprecipitation method with different lipid polymer nanoparticle formulation. NP KU55933 using PLGA formulation has the best loading efficacy as well as prolonged drug release profile. We demonstrated that NP KU55933 is a potent radiosensitizer in vitro using clonogenic assay and is more effective as a radiosensitizer than free KU55933 in vivo using mouse xenograft models of non-small cell lung cancer (NSCLC). Western blots and immunofluorescence showed NP KU55933 exhibited more prolonged inhibition of DNA repair pathway. In addition, NP KU55933 leads to lower skin toxicity than KU55933. Our study supports further investigations using NP to deliver DSBRIs to improve cancer radiotherapy treatment.

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project

PubMed Central

Benammar Elgaaied, Amel; Cascio, Donato; Bruno, Salvatore; Ciaccio, Maria Cristina; Cipolla, Marco; Fauci, Alessandro; Morgante, Rossella; Taormina, Vincenzo; Gorgi, Yousr; Marrakchi Triki, Raja; Ben Ahmed, Melika; Louzir, Hechmi; Yalaoui, Sadok; Imene, Sfar; Issaoui, Yassine; Abidi, Ahmed; Ammar, Myriam; Bedhiafi, Walid; Ben Fraj, Oussama; Bouhaha, Rym; Hamdi, Khouloud; Soumaya, Koudhi; Neili, Bilel; Asma, Gati; Lucchese, Mariano; Catanzaro, Maria; Barbara, Vincenza; Brusca, Ignazio; Fregapane, Maria; Amato, Gaetano; Friscia, Giuseppe; Neila, Trai; Turkia, Souayeh; Youssra, Haouami; Rekik, Raja; Bouokez, Hayet; Vasile Simone, Maria; Fauci, Francesco; Raso, Giuseppe

2016-01-01

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%). PMID:27042658

Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks.

PubMed

Wang, Huichen; Boecker, Wilfried; Wang, Hongyan; Wang, Xiang; Guan, Jun; Thompson, Larry H; Nickoloff, Jac A; Iliakis, George

2004-01-22

We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

Immunocytochemical localization of calretinin in the superficial layers of the cat superior colliculus.

PubMed

Hong, Soo-Kyung; Kim, Jee-Young; Jeon, Chang-Jin

2002-11-01

We localized calretinin-immunoreactive (IR) fibers and cells in the superior colliculus (SC) of the cat and studied the distribution and effect of enucleation on the distribution of this protein. Calretinin was localized with antibody immunocytochemistry. A dense plexus of anti-calretinin-IR fibers was found within the upper part of the superficial gray layer. Almost all of the labeled fibers were small diameter fibers with few varicosities. Monocular enucleation produced an almost complete reduction of calretinin-IR fibers in the SC contralateral to the enucleation. Furthermore, many calretinin-IR cells appeared in the contralateral SC. The newly appeared cells had small- to medium-sized vertical fusiform, oval or round, or stellate cell bodies. Two-color immunofluorescence revealed that no cells in the superficial layers expressed both calretinin and GABA. Many retinal ganglion cells were labeled after injections of retrograde axonal transport horseradish peroxidase (HRP) in the superficial layers. However, no large cells were double-labeled with calretinin and HRP. More than 95% of the double-labeled cells were small cells (<15 microm). Based on the retinal ganglion cell size, we believe that the vast majority of calretinin-IR retinocollicular fibers in cat SC are small gamma type cells that have W type physiologies.

Rickettsia sibirica mongolitimonae Infection, Turkey, 2016.

PubMed

Kuscu, Ferit; Orkun, Omer; Ulu, Aslihan; Kurtaran, Behice; Komur, Suheyla; Inal, A Seza; Erdogan, Damla; Tasova, Yesim; Aksu, Hasan S Z

2017-07-01

In 2016, Rickettsia sibirica mongolitimonae was diagnosed for a man in Turkey. He had been bitten by a Hyalomma marginatum tick, from which PCR detected rickettsial DNA. Sequence analysis of the DNA identified R. sibirica mongolitimonae. Immunofluorescence assay of patient serum indicated R. conorii, which cross-reacts. PCR is recommended for rickettsiosis diagnoses.

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

PubMed

Antico, A; Platzgummer, S; Bassetti, D; Bizzaro, N; Tozzoli, R; Villalta, D

2010-07-01

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism.

PubMed

Ba, Xiao-Hong; Min, Lian-Qiu

2015-01-01

The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P < 0.005). Apoptosis rate was significantly lower in EGb761-pretreated groups than in OGD group (P < 0.001). Immunofluorescent staining showed that there was AIF nuclear translocation in both EGb761-pretreated groups and OGD group, but AIF nuclear translocation was less in EGb761-pretreated groups than in OGD group. EGb761 can reduce the apoptosis of OGD SH-SY5Y cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice.

Pubertally born neurons and glia are functionally integrated into limbic and hypothalamic circuits of the male Syrian hamster.

PubMed

Mohr, Margaret A; Sisk, Cheryl L

2013-03-19

During puberty, the brain goes through extensive remodeling, involving the addition of new neurons and glia to brain regions beyond the canonical neurogenic regions (i.e., dentate gyrus and olfactory bulb), including limbic and hypothalamic cell groups associated with sex-typical behavior. Whether these pubertally born cells become functionally integrated into neural circuits remains unknown. To address this question, we gave male Syrian hamsters daily injections of the cell birthdate marker bromodeoxyuridine throughout puberty (postnatal day 28-49). Half of the animals were housed in enriched environments with access to a running wheel to determine whether enrichment increased the survival of pubertally born cells compared with the control environment. At 4 wk after the last BrdU injection, animals were allowed to interact with a receptive female and were then killed 1 h later. Triple-label immunofluorescence for BrdU, the mature neuron marker neuronal nuclear antigen, and the astrocytic marker glial fibrillary acidic protein revealed that a proportion of pubertally born cells in the medial preoptic area, arcuate nucleus, and medial amygdala differentiate into either mature neurons or astrocytes. Double-label immunofluorescence for BrdU and the protein Fos revealed that a subset of pubertally born cells in these regions is activated during sociosexual behavior, indicative of their functional incorporation into neural circuits. Enrichment affected the survival and activation of pubertally born cells in a brain region-specific manner. These results demonstrate that pubertally born cells located outside of the traditional neurogenic regions differentiate into neurons and glia and become functionally incorporated into neural circuits that subserve sex-typical behaviors.

Endothelial and smooth muscle cells from abdominal aortic aneurysm have increased oxidative stress and telomere attrition.

PubMed

Cafueri, Giuseppe; Parodi, Federica; Pistorio, Angela; Bertolotto, Maria; Ventura, Francesco; Gambini, Claudio; Bianco, Paolo; Dallegri, Franco; Pistoia, Vito; Pezzolo, Annalisa; Palombo, Domenico

2012-01-01

Abdominal aortic aneurysm (AAA) is a complex multi-factorial disease with life-threatening complications. AAA is typically asymptomatic and its rupture is associated with high mortality rate. Both environmental and genetic risk factors are involved in AAA pathogenesis. Aim of this study was to investigate telomere length (TL) and oxidative DNA damage in paired blood lymphocytes, aortic endothelial cells (EC), vascular smooth muscle cells (VSMC), and epidermal cells from patients with AAA in comparison with matched controls. TL was assessed using a modification of quantitative (Q)-FISH in combination with immunofluorescence for CD31 or α-smooth muscle actin to detect EC and VSMC, respectively. Oxidative DNA damage was investigated by immunofluorescence staining for 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG). Telomeres were found to be significantly shortened in EC, VSMC, keratinocytes and blood lymphocytes from AAA patients compared to matched controls. 8-oxo-dG immunoreactivity, indicative of oxidative DNA damage, was detected at higher levels in all of the above cell types from AAA patients compared to matched controls. Increased DNA double strand breaks were detected in AAA patients vs controls by nuclear staining for γ-H2AX histone. There was statistically significant inverse correlation between TL and accumulation of oxidative DNA damage in blood lymphocytes from AAA patients. This study shows for the first time that EC and VSMC from AAA have shortened telomeres and oxidative DNA damage. Similar findings were obtained with circulating lymphocytes and keratinocytes, indicating the systemic nature of the disease. Potential translational implications of these findings are discussed.

Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers.

PubMed

Onoyovwe, Akpevwe; Hagel, Jillian M; Chen, Xue; Khan, Morgan F; Schriemer, David C; Facchini, Peter J

2013-10-01

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.

Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

PubMed

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

2015-10-06

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

PubMed Central

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M.; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E.; Allen, Peter J.; Sempere, Lorenzo F.; Haab, Brian B.

2016-01-01

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis. PMID:26339978

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

PubMed Central

Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

The neurotoxicant, cuprizone, retards the differentiation of oligodendrocytes in vitro.

PubMed

Cammer, W

1999-10-15

The effects of oxalyldihydrazone (cuprizone) on weanling rodents provided an early protocol for toxic demyelination in vivo, in which degeneration of oligodendrocytes preceded disruption of the myelin sheath, and in which remyelination could take place. We administered cuprizone to oligodendrocyte-enriched glial-cell cultures and to mixed glial-cell cultures from neonatal rat brains. The cultures were treated with cuprizone for 1 h and allowed to continue differentiating on subsequent days. Treated cultures and respective control cultures were fixed with 4% paraformaldehyde (w/v) and immunostained with double immunofluorescence. MAbO4 was used to mark precursors and mature oligodendrocytes, and anti-myelin basic protein (MBP) to mark mature oligodendrocytes (O4+/MBP+), as distinguished from precursors, which were O4+/MBP-. Cell counts suggested that cuprizone inhibited the maturation of oligodendrocytes without diminishing the numbers of precursors, and appeared to affect the mitochondria in those cells.

Profiles of gonadotropin-inhibitory hormone and melatonin during the sex change and maturation of cinnamon clownfish, Amphiprion melanopus.

PubMed

Choi, Young Jae; Habibi, Hamid R; Choi, Cheol Young

2016-06-24

The present study aimed to determine the relationship between melatonin and gonadotropin-inhibitory hormone (GnIH) and their effect on reproduction in cinnamon clownfish, Amphiprion melanopus. Accordingly, we investigated the expression pattern of GnIH, GnIH receptor (GnIH-R), and melatonin receptor (MT-R1) mRNA and protein, as well as the plasma levels of melatonin, during sex change in cinnamon clownfish. We found that GnIH and MT-R1 mRNA and melatonin activity were higher in fish with mature brain than in fish with developing gonads, and using double immunofluorescence staining, we found that both GnIH and MT-R1 proteins were co-expressed in the hypothalamus of cinnamon clownfish. These findings support the hypothesis that melatonin plays an important role in the negative regulation of maturation and GnIH regulation during reproduction. Copyright © 2016 Elsevier Inc. All rights reserved.

Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins.

PubMed

Ranz, A I; Miguet, J G; Anaya, C; Venteo, A; Cortés, E; Vela, C; Sanz, A

1992-11-01

A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.

Long-Term Survival and Intracellular Replication of Mycoplasma hominis in Trichomonas vaginalis Cells: Potential Role of the Protozoon in Transmitting Bacterial Infection

PubMed Central

Dessì, Daniele; Delogu, Giuseppe; Emonte, Eleonora; Catania, Maria Rosaria; Fiori, Pier Luigi; Rappelli, Paola

2005-01-01

The existence of a symbiotic relationship between Trichomonas vaginalis and Mycoplasma hominis, which is the first reported example of symbiosis between two obligate human pathogens, has been recently reported by our research group. In this work, we examined the cellular location of M. hominis in respect to T. vaginalis. By using gentamicin protection assays, double immunofluorescence, and confocal microscopy, we obtained strong evidence that M. hominis is located within protozoan cells. 5-Bromodeoxyuridine incorporation assays showed that intracellularly located mycoplasmas actively synthesize DNA. Our results demonstrate that M. hominis has the capability of entering trichomonad cells and of replicating inside the protozoon. These findings suggest that symbiosis might provide the bacteria, during human infection, with the capability to resist to environmental stresses, such as host defense mechanisms and pharmacological therapies. PMID:15664961

Thymic hormone-containing cells. Characterization and localization of serum thymic factor in young mouse thymus studied by monoclonal antibodies

PubMed Central

1982-01-01

The characterization and distribution of cells containing the serum thymic factor (FTS) in the thymus of young mice was studied by immunofluorescence using monoclonal anti-FTS antibodies. FTS+ cells were distributed throughout the thymic parenchyma but were more frequent in the medullary region than in the cortex. FTS-containing cells presented a stellate or globular aspect, and some of them exhibited fluorescent cytoplasmic granules. The epithelial nature of FTS+ cells was confirmed by double-labeling experiments using an anti- keratin antiserum (as an epithelial cell marker). Nevertheless, only a minority of keratin-positive epithelial reticular cells contained FTS. All controls, including the incubation of sections from nonthymic tissues with the anti-FTS antibodies, were negative. Taken together, these results confirm the exclusive localization of FTS-containing cells within the mouse thymus. PMID:7047671

Thymic hormone containing cells. II. Evolution of cells containing the serum thymic factor (FTS or thymulin) in normal and autoimmune mice, as revealed by anti-FTS monoclonal antibodies. Relationship with Ia bearing cells.

PubMed Central

Savino, W; Dardenne, M; Bach, J F

1983-01-01

The number of thymic epithelial cells containing the serum thymic factor (FTS or thymulin), assessed by indirect immunofluorescence using an anti-FTS monoclonal antibody, was studied in the thymus of normal and autoimmune mice as a function of age. In normal mice the number of FTS+ cells was constant until the age of 6 months and then began to decline. In autoimmune strains, the age linked decline was premature being already significant at 10 weeks of age. These findings were paralleled by the age associated decline of FTS blood levels in all strains studied. Double labelling experiments showed that in both normal and autoimmune mice, FTS+ cells were Ia negative, suggesting that these cells belong to a specific subpopulation of the thymic epithelial reticulum. PMID:6345030

[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

PubMed

Rafalovskaia-Orlovskaia, E P; Gorgidze, L A; Gladkikh, A A; Tauger, S M; Vorob'ev, I A

2012-01-01

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins.more » Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.« less

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication

PubMed Central

Kim, Ja Yeon; Wang, Linya; Lee, Jiyoung

2017-01-01

ABSTRACT Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication. IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells. PMID:28747506

Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

PubMed

Mian, Elisabeth; Wiesmüller, Lisa

2017-01-01

Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells treated with and without pharmacological kinase inhibitors revealed ATM-dependent chromatin remodeling at the MLL break site giving access to DNA repair proteins but also nucleases triggering MLL rearrangements. This chapter summarizes these methods for functional characterization of ATM in patient LCLs and human cell lines.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

Immunofluorescence Staining Protocols for Major Autophagy Proteins Including LC3, P62, and ULK1 in Mammalian Cells in Response to Normoxia and Hypoxia.

PubMed

Li, Wen; Li, Shupeng; Li, Yifang; Lin, Xiaoying; Hu, Yongquan; Meng, Tian; Wu, Baojin; He, Rongrong; Feng, Du

2018-03-27

Immunofluorescence is an invaluable technique widely used in cell biology. This technique allows visualization of the subcellular distribution of different target proteins or organelles, by specific recognition of the antibody to the endogenous protein itself or to its antigen via the epitope. This technique can be used on tissue sections, cultured cells, or individual cells. Meanwhile, immunofluorescence can also be used in combination with non-antibody fluorescent staining, such as DAPI or fluorescent fusion proteins, e.g., GFP or YFP, etc.Autophagy is a catabolic pathway in which dysfunctional organelles and cellular components are degraded via lysosomes. During this process, cytoplasmic LC3 translocates to autophagosomal membranes. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 or other autophagy markers. Immunofluorescence is an important part of autophagy detection methods even if observation of the formation of autophagosome by transmission electron microscopy has become a gold standard for characterizing autophagy.By observing the immunofluorescence staining of some key autophagy proteins, we can intuitively evaluate the levels of autophagy in samples. Herein, this protocol describes the predominant method used for the research of autophagy, which mainly focuses on the immunofluorescence staining of cellular LC3, P62, and ULK1 in response to normoxia and hypoxia, by presenting the detailed materials required and methodology.

Rickettsia sibirica mongolitimonae Infection, Turkey, 2016

PubMed Central

Orkun, Omer; Ulu, Aslihan; Kurtaran, Behice; Komur, Suheyla; Inal, A. Seza; Erdogan, Damla; Tasova, Yesim; Aksu, Hasan S.Z.

2017-01-01

In 2016, Rickettsia sibirica mongolitimonae was diagnosed for a man in Turkey. He had been bitten by a Hyalomma marginatum tick, from which PCR detected rickettsial DNA. Sequence analysis of the DNA identified R. sibirica mongolitimonae. Immunofluorescence assay of patient serum indicated R. conorii, which cross-reacts. PCR is recommended for rickettsiosis diagnoses. PMID:28628458

Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma

PubMed Central

Becker, Aline; Elder, Brad; Puduvalli, Vinay; Winter, Jessica; Gurcan, Metin

2017-01-01

Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology. PMID:28282372

A light therapy for treating Alzheimer's disease

NASA Astrophysics Data System (ADS)

Wang, Xue; Han, Mengmeng; Wang, Qiyan; Zeng, Yuhui; Meng, Qingqiang; Zhang, Jun; Wei, Xunbin

2017-02-01

It is generally believed that there are some connections between Alzheimer's disease and amyloid protein plaques in the brain. The typical symptoms of Alzheimer's disease are memory loss, language disorders, mood swings, loss of motivation and behavioral issues. Currently, the main therapeutic method is pharmacotherapy, which may temporarily reduce symptoms, but has many side effects. Infrared light therapy has been studied in a range of single and multiple irradiation protocols in previous studies and was found beneficial for neuropathology. In our research we have studied the effect of infrared light on Alzheimer's disease through transgenic mouse model. We designed an experimental apparatus for treating mice, which primarily included a therapeutic box and a LED array, which emitted infrared light. After the treatment, we assessed the effects of infrared light by performing two tests: cognitive performance of mice in Morris water maze, and plaque load by immunofluorescence analysis. Immunofluorescence analysis was based on measuring the quantity of plaques in mouse brain slices. Our results show that infrared therapy is able to improve cognitive performance in the mouse model. It might provide a novel and safe way to treat Alzheimer's disease.

Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin.

PubMed

Wu, Chung-Chun; Fang, Chih-Yeu; Cheng, Yu-Jhen; Hsu, Hui-Yu; Chou, Sheng-Ping; Huang, Sheng-Yen; Tsai, Ching-Hwa; Chen, Jen-Yang

2017-01-05

Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.

Immunofluorescent localization of Staphylococcos aureus antigen in acute bacterial endocarditis nephritis.

PubMed

Yum, M; Wheat, L J; Maxwell, D; Edwards, J L

1978-11-01

A 75-year-old man with Staphylococcus aureus endocarditis in whom acute diffuse proliferative glomerulonephritis developed is described. The light- and electron-microscopic changes of the glomeruli in this case were identical to those of acute poststreptococcal glomerulonephritis. Immunofluorescence revealed deposition of immunoglobulins and complement in the glomeruli. In addition, bacterial antigenic material was demonstrated in the glomeruli by indirect immunofluorescence. These observations further support the hypothesis of an immune-complex pathogenesis in this form of glomerulonephritis.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

PubMed

Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

2015-03-08

Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

PubMed Central

1978-01-01

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis. PMID:102651

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase.

PubMed

Ramírez-Mata, Alberto; Michalak, Colette; Mendoza-Hernández, Guillermo; León-Del-Río, Alfonso; González-Noriega, Alfonso

2011-10-01

Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase. Copyright © 2011 Elsevier Inc. All rights reserved.

Bile duct regeneration and immune response by passenger lymphocytes signals biliary recovery versus complications after liver transplantation.

PubMed

Junger, Henrik H; Schlitt, Hans J; Geissler, Edward K; Fichtner-Feigl, Stefan; Brunner, Stefan M

2017-11-01

This study aimed to elucidate the impact of epithelial regenerative responses and immune cell infiltration on biliary complications after liver transplantation. Bile duct (BD) damage after cold storage was quantified by a BD damage score and correlated with patient outcome in 41 patients. Bacterial infiltration was determined by fluorescence in situ hybridization (FISH). BD samples were analyzed by immunohistochemistry for E-cadherin, cytokeratin, CD56, CD14, CD4, CD8, and double-immunofluorescence for cytokine production and by messenger RNA (mRNA) microarray. Increased mRNA levels of adherens junctions (P < 0.01) were detected in damaged BDs from patients without complications compared with damaged BDs from patients with biliary complications. Immunohistochemistry showed increased expression of E-cadherin and cytokeratin in BDs without biliary complications (P = 0.03; P = 0.047). FISH analysis demonstrated translocation of bacteria in BDs. However, mRNA analysis suggested an enhanced immune response in BDs without biliary complications (P < 0.01). Regarding immune cell infiltration, CD4 + and CD8 + cells were significantly increased in patients without complications compared with those with complications (P = 0.02; P = 0.01). In conclusion, following BD damage during cold storage, we hypothesize that the functional regenerative capacity of biliary epithelium and enhanced local adaptive immune cell infiltration are crucial for BD recovery. Such molecular immunological BD analyses therefore could help to predict biliary complications in cases of "major" epithelial damage after cold storage.Liver Transplantation 23 1422-1432 2017 AASLD. © 2017 by the American Association for the Study of Liver Diseases.

Immunohistochemical detection of intrathrombotic fibrocytes and its application to thrombus age estimation in murine deep vein thrombosis model.

PubMed

Nosaka, Mizuho; Ishida, Yuko; Kimura, Akihiko; Kawaguchi, Takashi; Yamamoto, Hiroki; Kuninaka, Yumi; Kondo, Toshikazu

2017-01-01

Deep vein thrombi are dissolved after fibrosis process along with an increase of thrombus age. Fibrocytes are circulating bone marrow-derived cells with mesenchymal features that potentially have a unique and critical function in fibrosis. In this study, a double-color immunofluorescence analysis was carried out by using anti-CD45 and anti-collagen type I antibodies to examine the time-dependent appearance of fibrocytes in the murine model of stasis-induced deep vein thrombosis. The thrombus ages were 1, 3, 5, 7, 10, 14, and 21 days. In a thrombus age of less than 5 days, CD45 + and collagen type I + fibrocytes were never detected. The intrathrombotic fibrocytes were initially observed in thrombi aged 7 days, and their number increased with advances in thrombus age. In a quantitative morphometrical analysis, the average number of intrathrombotic fibrocytes was highest in 14-day-old thrombi, and all of the five samples aged 14 days had the fibrocyte number of more than 25, and in three out of them, the number of intrathrombotic fibrocytes was over 30. On the contrary, in all of thrombus samples with the postligation intervals of 10 and 21 days, the number of intrathrombotic fibrocytes was less than 25. These observations imply that thrombi containing fibrocytes are at least 7 days old and that a fibrocyte number exceeding 30 would indicate the thrombus age of approximately 14 days. Our observations indicate that the detection of fibrocytes could be useful for thrombus age determination.

Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo

PubMed Central

Moyen, Nanikaly; Thirion, Laurence; Emmerich, Petra; Dzia-Lepfoundzou, Amelia; Richet, Hervé; Boehmann, Yannik; Dimi, Yannick; Gallian, Pierre; Gould, Ernest A.; Günther, Stephan; de Lamballerie, Xavier

2015-01-01

Background Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV. Methodology and Findings In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds. Conclusions/Significance This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors. PMID:26047124

Sequential Immunofluorescent Light Microscopy and Electron Microscopy of Recombination Nodules During Meiotic Prophase I.

PubMed

Anderson, Lorinda K

2017-01-01

Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers.

PubMed

Terraneo, Laura; Bianciardi, Paola; Malavalli, Ashok; Mkrtchyan, Gnel; Spann, Stephanie N; Lohman, Jeff; Samaja, Michele; Vandegriff, Kim D

2017-06-01

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Electrostatic association of glutathione transferase to the nuclear membrane. Evidence of an enzyme defense barrier at the nuclear envelope.

PubMed

Stella, Lorenzo; Pallottini, Valentina; Moreno, Sandra; Leoni, Silvia; De Maria, Francesca; Turella, Paola; Federici, Giorgio; Fabrini, Raffaele; Dawood, Kutayba F; Bello, Mario Lo; Pedersen, Jens Z; Ricci, Giorgio

2007-03-02

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.

Regional Myelin and Axon Damage and Neuroinflammation in the Adult Mouse Brain After Long-Term Postnatal Vanadium Exposure.

PubMed

Azeez, Idris A; Olopade, Funmilayo; Laperchia, Claudia; Andrioli, Anna; Scambi, Ilaria; Onwuka, Silas K; Bentivoglio, Marina; Olopade, James O

2016-09-01

Environmental exposure to vanadium occurs in areas of persistent burning of fossil fuels; this metal is known to induce oxidative stress and oligodendrocyte damage. Here, we determined whether vanadium exposure (3 mg/kg) in mice during the first 3 postnatal months leads to a sustained neuroinflammatory response. Body weight monitoring, and muscle strength and open field tests showed reduction of body weight gain and locomotor impairment in vanadium-exposed mice. Myelin histochemistry and immunohistochemistry for astrocytes, microglia, and nonphosphorylated neurofilaments revealed striking regional heterogeneity. Myelin damage involved the midline corpus callosum and fibers in cortical gray matter, hippocampus, and diencephalon that were associated with axonal damage. Astrocyte and microglial activation was identified in the same regions and in the internal capsule; however, no overt myelin and axon damage was observed in the latter. Double immunofluorescence revealed induction of high tumor necrosis factor (TNF) immunoreactivity in reactive astrocytes. Western blotting analysis showed significant induction of TNF and interleukin-1β expression. Together these findings show that chronic postnatal vanadium exposure leads to functional deficit and region-dependent myelin damage that does not spare axons. This injury is associated with glial cell activation and proinflammatory cytokine induction, which may reflect both neurotoxic and neuroprotective responses. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

A Link between Meiotic Prophase Progression and CrossoverControl

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlton, Peter M.; Farruggio, Alfonso P.; Dernburg, Abby F.

2005-07-06

During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealedmore » that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency.« less

P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro.

PubMed

Heine, C; Heimrich, B; Vogt, J; Wegner, A; Illes, P; Franke, Heike

2006-01-01

Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5'-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro, double immunofluorescence labeling revealed the expression of P2X(1), P2X(2), P2X(4) as well as P2Y(1) and P2Y(2) receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5'-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.

Dopamine synthesis and dopamine receptor expression are disturbed in recurrent miscarriages

PubMed Central

Gratz, Michael J; Stavrou, Stavroula; Kuhn, Christina; Hofmann, Simone; Hermelink, Kerstin; Heidegger, Helene; Hutter, Stefan; Mayr, Doris; Mahner, Sven; Jeschke, Udo; Vattai, Aurelia

2018-01-01

Objectives l-dopa decarboxylase (DDC) is responsible for the synthesis of dopamine. Dopamine, which binds to the D2-dopamine receptor (D2R), plays an important role in the maintenance of pregnancy. Aim of our study was the analysis of DDC and D2R expression in placentas of spontaneous miscarriages (SMs) and recurrent miscarriages (RMs) in comparison to healthy controls. Methods Patients with SM (n = 15) and RM (n = 15) were compared with patients from healthy pregnancies (n = 15) (pregnancy weeks 7–13 each). Placental tissue has been collected from SMs and RMs from the first trimester (Department of Gynaecology and Obstetrics, LMU Munich) and from abruptions (private practice, Munich). Placental cell lines, BeWo- and JEG-3 cells, were stimulated with the trace amines T0AM and T1AM in vitro. Results Levels of DDC and D2R in trophoblasts and the decidua were lower in RMs in comparison to healthy controls. Stimulation of BeWo cells with T1AM significantly reduced DDC mRNA and protein levels. Via double-immunofluorescence, a DDC-positive cell type beneath decidual stromal cells and foetal EVT in the decidua could be detected. Conclusions Downregulation of DDC and D2R in trophoblasts of RMs reflects a reduced signal cascade of catecholamines on the foetal side. PMID:29686031

Dopamine synthesis and dopamine receptor expression are disturbed in recurrent miscarriages.

PubMed

Gratz, Michael J; Stavrou, Stavroula; Kuhn, Christina; Hofmann, Simone; Hermelink, Kerstin; Heidegger, Helene; Hutter, Stefan; Mayr, Doris; Mahner, Sven; Jeschke, Udo; Vattai, Aurelia

2018-05-01

l-dopa decarboxylase (DDC) is responsible for the synthesis of dopamine. Dopamine, which binds to the D 2 -dopamine receptor (D2R), plays an important role in the maintenance of pregnancy. Aim of our study was the analysis of DDC and D2R expression in placentas of spontaneous miscarriages (SMs) and recurrent miscarriages (RMs) in comparison to healthy controls. Patients with SM (n = 15) and RM (n = 15) were compared with patients from healthy pregnancies (n = 15) (pregnancy weeks 7-13 each). Placental tissue has been collected from SMs and RMs from the first trimester (Department of Gynaecology and Obstetrics, LMU Munich) and from abruptions (private practice, Munich). Placental cell lines, BeWo- and JEG-3 cells, were stimulated with the trace amines T 0 AM and T 1 AM in vitro . Levels of DDC and D2R in trophoblasts and the decidua were lower in RMs in comparison to healthy controls. Stimulation of BeWo cells with T 1 AM significantly reduced DDC mRNA and protein levels. Via double-immunofluorescence, a DDC-positive cell type beneath decidual stromal cells and foetal EVT in the decidua could be detected. Downregulation of DDC and D2R in trophoblasts of RMs reflects a reduced signal cascade of catecholamines on the foetal side. © 2018 The authors.

Phospho-Tau Accumulation and Structural Alterations of the Golgi Apparatus of Cortical Pyramidal Neurons in the P301S Tauopathy Mouse Model

PubMed Central

Antón-Fernández, Alejandro; Merchán-Rubira, Jesús; Avila, Jesús; Hernández, Félix; DeFelipe, Javier; Muñoz, Alberto

2017-01-01

The Golgi apparatus (GA) is a highly dynamic organelle involved in the processing and sorting of cellular proteins. In Alzheimer’s disease (AD), it has been shown to decrease in size and become fragmented in neocortical and hippocampal neuronal subpopulations. This fragmentation and decrease in size of the GA in AD has been related to the accumulation of hyperphosphorylated tau. However, the involvement of other pathological factors associated with the course of the disease, such as the extracellular accumulation of amyloid-β (Aβ) aggregates, cannot be ruled out, since both pathologies are present in AD patients. Here we use the P301S tauopathy mouse model to examine possible alterations of the GA in neurons that overexpress human tau (P301S mutated gene) in neocortical and hippocampal neurons, using double immunofluorescence techniques and confocal microscopy. Quantitative analysis revealed that neurofibrillary tangle (NFT)-bearing neurons had important morphological alterations and reductions in the surface area and volume of the GA compared with NFT-free neurons. Since in this mouse model there are no Aβ aggregates typical of AD, the present findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA, and that these changes may occur in the absence of Aβ pathology. PMID:28922155

Phospho-Tau Accumulation and Structural Alterations of the Golgi Apparatus of Cortical Pyramidal Neurons in the P301S Tauopathy Mouse Model.

PubMed

Antón-Fernández, Alejandro; Merchán-Rubira, Jesús; Avila, Jesús; Hernández, Félix; DeFelipe, Javier; Muñoz, Alberto

2017-01-01

The Golgi apparatus (GA) is a highly dynamic organelle involved in the processing and sorting of cellular proteins. In Alzheimer's disease (AD), it has been shown to decrease in size and become fragmented in neocortical and hippocampal neuronal subpopulations. This fragmentation and decrease in size of the GA in AD has been related to the accumulation of hyperphosphorylated tau. However, the involvement of other pathological factors associated with the course of the disease, such as the extracellular accumulation of amyloid-β (Aβ) aggregates, cannot be ruled out, since both pathologies are present in AD patients. Here we use the P301S tauopathy mouse model to examine possible alterations of the GA in neurons that overexpress human tau (P301S mutated gene) in neocortical and hippocampal neurons, using double immunofluorescence techniques and confocal microscopy. Quantitative analysis revealed that neurofibrillary tangle (NFT)-bearing neurons had important morphological alterations and reductions in the surface area and volume of the GA compared with NFT-free neurons. Since in this mouse model there are no Aβ aggregates typical of AD, the present findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA, and that these changes may occur in the absence of Aβ pathology.

The effect of CXCR2 inhibition on seizure activity in the pilocarpine epilepsy mouse model.

PubMed

Xu, Tao; Yu, Xinyuan; Wang, Teng; Liu, Ying; Liu, Xi; Ou, Shu; Chen, Yangmei

2017-09-01

C-X-C motif chemokine receptor 2 (CXCR2) is one of the most well characterized chemokine receptors and is a potential target for treating brain pathologies involving inflammatory processes, including epilepsy. However, the role of CXCR2 in epilepsy has not been investigated, and whether CXCR2 modulates seizure activity in temporal lobe epilepsy (TLE) remains unknown. In this study, we aimed to determine the potential role of CXCR2 in intractable TLE patients and in pilocarpine-induced epileptic mice. Here, through Western blotting and semi-quantitative immunohistochemistry, we detected that CXCR2 protein expression was up-regulated (by nearly 50%) in the temporal neocortex of TLE patients and in the hippocampus and adjacent temporal cortex of pilocarpine mice model. Double-label immunofluorescence and immunohistochemical analysis indicated that CXCR2 was expressed in neurons. To investigate the effect of the CXCR2 selective antagonist SB225002 on seizure activity, SB225002 was i.p. administered during the latency window of spontaneous recurrent seizures (SRSs). This treatment increased (by nearly 40%) the latency of SRSs and reduced (by nearly 50%) the frequency of SRSs during the chronic period of epilepsy. This study suggests that CXCR2 plays a critical role in modifying epileptic seizure activity and that CXCR2 blockade could be a potential molecular therapeutic target for epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression and Significance of Neuroligins in Myenteric Cells of Cajal in Hirschsprung's Disease

PubMed Central

Wang, Jian; Mou, Yaru; Zhang, Qiangye; Zhang, Fan; Yang, Hongchao; Zhang, Wentong; Li, Aiwu

2013-01-01

Purpose The aim of this study was to investigate the expression and significance of neuroligins in myenteric cells of Cajal (ICC-MY) in Hirschsprung’s disease (HSCR). Methods Longitudinal muscle with adherent myenteric plexus (LMMP) from surgical excision waste colon of HSCR children were prepared by peeling off the mucous layer, sub-mucosal layer and circular muscle. Neuroligins, c-Kit (c-Kit-immunoreactivity representing ICC) and their relationship were assessed by double labeling immunofluorescence staining. ICC-MY were dissociated and cultured from LMMP by enzymolysis method, and were purified and analyzed using a combination of magnetic-activated cell sorting (MACS) and flow cytometry (FCM). Western-blot analysis was applied to compare and evaluate the expression levels of neuroligins in ICC-MY which were dissociated from different segments of HSCR (ganglionic colonic segment, transitional colonic segment and aganglionic colonic segment). Results Neuroligins and c-Kit were expressed on the same cells (ICC-MY); ICC-MY were dissociated, cultured and purified. For HSCR, neuroligins were expressed significantly in ICC-MY from ganglionic colonic segments, moderately in those from transitional colonic segments and down-regulated significantly in those from aganglionic colonic segments. Conclusions Neuroligins were expressed in ICC-MY of human beings, and the expression varies from different segments of HSCR. This abnormal expression might play an important role in the pathogenesis of this disease through affecting the synaptic function of ICC-MY. PMID:23840625

The RNA-editing deaminase ADAR is involved in stress resistance of Artemia diapause embryos.

PubMed

Dai, Li; Liu, Xue-Chen; Ye, Sen; Li, Hua-Wei; Chen, Dian-Fu; Yu, Xiao-Jian; Huang, Xue-Ting; Zhang, Li; Yang, Fan; Yang, Jin-Shu; Yang, Wei-Jun

2016-11-01

The most widespread type of RNA editing, conversion of adenosine to inosine (A→I), is catalyzed by two members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2. These enzymes edit transcripts for neurotransmitter receptors and ion channels during adaption to changes in the physical environment. In the primitive crustacean Artemia, when maternal adults are exposed to unfavorable conditions, they release diapause embryos to withstand harsh environments. The aim of the current study was therefore to elucidate the role of ADAR of Artemia diapause embryos in resistance to stress. Here, we identified Artemia ADAR (Ar-ADAR), which harbors a putative nuclear localization sequence (NLS) and two double-stranded RNA-binding motifs (dsRBMs) in the amino-terminal region and an adenosine deaminase (AD) domain in the carboxyl-terminal region. Western blot and immunofluorescence analysis revealed that Ar-ADAR is expressed abundantly in post-diapause embryos. Artemia (n = 200, three replicates) were tested under basal and stress conditions. We found that Ar-ADAR was significantly induced in response to the stresses of salinity and heat-shock. Furthermore, in vivo knockdown of Ar-ADAR (n = 100, three replicates) by RNA interference induced formation of pseudo-diapause embryos, which lack resistance to the stresses and exhibit high levels of apoptosis. These results indicate that Ar-ADAR contributes to resistance to stress in Artemia diapause embryos.

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

PubMed

Suntharalingam, Saravanabavaan; Mladenov, Emil; Sarabhai, Theresia; Wetter, Axel; Kraff, Oliver; Quick, Harald H; Forsting, Michael; Iliakis, Georg; Nassenstein, Kai

2018-05-01

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, γ-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes. © RSNA, 2018.

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

PubMed

Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

2016-04-11

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

Core Histones and HIRIP3, a Novel Histone-Binding Protein, Directly Interact with WD Repeat Protein HIRA

PubMed Central

Lorain, Stéphanie; Quivy, Jean-Pierre; Monier-Gavelle, Frédérique; Scamps, Christine; Lécluse, Yann; Almouzni, Geneviève; Lipinski, Marc

1998-01-01

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development. PMID:9710638

A Subset of Palisade Endings Only in the Medial and Inferior Rectus Muscle in Monkey Contain Calretinin

PubMed Central

Lienbacher, Karoline; Ono, Seiji; Fleuriet, Jérome; Mustari, Michael; Horn, Anja K. E.

2018-01-01

Purpose To further chemically characterize palisade endings in extraocular muscles in rhesus monkeys. Methods Extraocular muscles of three rhesus monkeys were studied for expression of the calcium-binding protein calretinin (CR) in palisade endings and multiple endings. The complete innervation was visualized with antibodies against the synaptosomal-associated protein of 25 kDa and combined with immunofluorescence for CR. Six rhesus monkeys received tracer injections of choleratoxin subunit B or wheat germ agglutinin into either the belly or distal myotendinous junction of the medial or inferior rectus muscle to allow retrograde tracing in the C-group of the oculomotor nucleus. Double-immunofluorescence methods were used to study the CR content in retrogradely labeled neurons in the C-group. Results A subgroup of palisade and multiple endings was found to express CR, only in the medial and inferior rectus muscle. In contrast, the en plaque endings lacked CR. Accordingly, within the tracer-labeled neurons of the C-group, a subgroup expressed CR. Conclusions The study indicates that two different neuron populations targeting nontwitch muscle fibers are present within the C-group for inferior rectus and medial rectus, respectively, one expressing CR, one lacking CR. It is possible that the CR-negative neurons represent the basic population for all extraocular muscles, whereas the CR-positive neurons giving rise to CR-positive palisade endings represent a specialized, perhaps more excitable type of nerve ending in the medial and inferior rectus muscles, being more active in vergence. The malfunction of this CR-positive population of neurons that target nontwitch muscle fibers could play a significant role in strabismus.

Apple S-RNase interacts with an actin-binding protein, MdMVG, to reduce pollen tube growth by inhibiting its actin-severing activity at the early stage of self-pollination induction.

PubMed

Yang, Qing; Meng, Dong; Gu, Zhaoyu; Li, Wei; Chen, Qiuju; Li, Yang; Yuan, Hui; Yu, Jie; Liu, Chunsheng; Li, Tianzhong

2018-04-18

In S-RNase-mediated self-incompatibility, S-RNase secreted from the style destroys the actin cytoskeleton of the self-pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S-RNase does not bind or sever filamentous actin (F-actin). In apple (Malus domestica), we identified an actin-binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S-RNase and directly binds and severs F-actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S-RNase inhibits the F-actin-severing activity of MdMVG in vitro. In vivo, the addition of S-RNase to self-pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self-pollination induction in the presence of MdMVG overexpression. By generating 25 single-, double- and triple-point mutations in the amino acid motif E-E-K-E-K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple-point mutant, MdMVG (E167A/E171A/K185A) , that no longer has F-actin-severing activity or interacts with any of the four S-haplotype S-RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S-RNases. We conclude that apple S-RNase interacts with MdMVG to reduce self-pollen tube growth by inhibiting its F-actin-severing activity. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Involvement of hypothalamus autoimmunity in patients with autoimmune hypopituitarism: role of antibodies to hypothalamic cells.

PubMed

De Bellis, A; Sinisi, A A; Pane, E; Dello Iacovo, A; Bellastella, G; Di Scala, G; Falorni, A; Giavoli, C; Gasco, V; Giordano, R; Ambrosio, M R; Colao, A; Bizzarro, A; Bellastella, A

2012-10-01

Antipituitary antibodies (APA) but not antihypothalamus antibodies (AHA) are usually searched for in autoimmune hypopituitarism. Our objective was to search for AHA and characterize their hypothalamic target in patients with autoimmune hypopituitarism to clarify, on the basis of the cells stained by these antibodies, the occurrence of autoimmune subclinical/clinical central diabetes insipidus (CDI) and/or possible joint hypothalamic contribution to their hypopituitarism. We conducted a cross-sectional cohort study. Ninety-five APA-positive patients with autoimmune hypopituitarism, 60 without (group 1) and 35 with (group 2) lymphocytic hypophysitis, were studied in comparison with 20 patients with postsurgical hypopituitarism and 50 normal subjects. AHA by immunofluorescence and posterior pituitary function were evaluated; then AHA-positive sera were retested by double immunofluorescence to identify the hypothalamic cells targeted by AHA. AHA were detected at high titer in 12 patients in group 1 and in eight patients in group 2. They immunostained arginine vasopressin (AVP)-secreting cells in nine of 12 in group 1 and in four of eight in group 2. All AVP cell antibody-positive patients presented with subclinical/clinical CDI; in contrast, four patients with GH/ACTH deficiency but with APA staining only GH-secreting cells showed AHA targeting CRH- secreting cells. The occurrence of CDI in patients with lymphocytic hypophysitis seems due to an autoimmune hypothalamic involvement rather than an expansion of the pituitary inflammatory process. To search for AVP antibody in these patients may help to identify those of them prone to develop an autoimmune CDI. The detection of AHA targeting CRH-secreting cells in some patients with GH/ACTH deficiency but with APA targeting only GH-secreting cells indicates that an autoimmune aggression to hypothalamus is jointly responsible for their hypopituitarism.

Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

PubMed

Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2014-07-01

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Anti-pituitary antibodies against corticotrophs in IgG4-related hypophysitis.

PubMed

Iwata, Naoko; Iwama, Shintaro; Sugimura, Yoshihisa; Yasuda, Yoshinori; Nakashima, Kohtaro; Takeuchi, Seiji; Hagiwara, Daisuke; Ito, Yoshihiro; Suga, Hidetaka; Goto, Motomitsu; Banno, Ryoichi; Caturegli, Patrizio; Koike, Teruhiko; Oshida, Yoshiharu; Arima, Hiroshi

2017-06-01

IgG4-related disease is a systemic inflammatory disease characterized by infiltration of IgG4-positive plasma cells into multiple organs, including the pituitary gland. Autoimmunity is thought to be involved in the pathogenesis of IgG4-related disease. The diagnosis of IgG4-related hypophysitis (IgG4-RH) is difficult because its clinical features, such as pituitary swelling and hypopituitarism, are similar to those of other pituitary diseases, including lymphocytic hypophysitis and sellar/suprasellar tumors. The presence and significance of anti-pituitary antibodies (APA) in IgG4-RH is unclear. In this case-control study, we used single indirect immunofluorescence on human pituitary substrates to assess the prevalence of serum APA in 17 patients with IgG4-RH, 8 control patients with other pituitary diseases (lymphocytic infundibulo-neurohypophysitis, 3; craniopharyngioma, 2; germinoma, 3), and 9 healthy subjects. We further analyzed the endocrine cells targeted by the antibodies using double indirect immunofluorescence. APA were found in 5 of 17 patients with IgG4-RH (29%), and in none of the pituitary controls or healthy subjects. The endocrine cells targeted by the antibodies in the 5 IgG4-RH cases were exclusively corticotrophs. Antibodies were of the IgG1 subclass, rather than IgG4, in all 5 cases, suggesting that IgG4 is not directly involved in the pathogenesis. Finally, antibodies recognized pro-opiomelanocortin in 2 of the cases. Our study suggests that autoimmunity is involved in the pathogenesis of IgG4-RH and that corticotrophs are the main antigenic target, highlighting a possible new diagnostic marker for this condition.

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study.

PubMed

Myöhänen, T T; Venäläinen, J I; Garcia-Horsman, J A; Männistö, P T

2008-06-02

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism

PubMed Central

Ba, Xiao-Hong; Min, Lian-Qiu

2015-01-01

Objective: The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. Materials and Methods: SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Results: Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P < 0.005). Apoptosis rate was significantly lower in EGb761-pretreated groups than in OGD group (P < 0.001). Immunofluorescent staining showed that there was AIF nuclear translocation in both EGb761-pretreated groups and OGD group, but AIF nuclear translocation was less in EGb761-pretreated groups than in OGD group. Conclusion: EGb761 can reduce the apoptosis of OGD SH-SY5Y cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice. PMID:25821320

Preprodynorphin-expressing neurons constitute a large subgroup of somatostatin-expressing GABAergic interneurons in the mouse neocortex.

PubMed

Sohn, Jaerin; Hioki, Hiroyuki; Okamoto, Shinichiro; Kaneko, Takeshi

2014-05-01

Dynorphins, leumorphin, and neoendorphins are preprodynorphin (PPD)-derived peptides and ligands for κ-opioid receptors. Using an antibody to PPD C-terminal, we investigated the chemical and molecular characteristics of PPD-expressing neurons in mouse neocortex. PPD-immunopositive neuronal somata were distributed most frequently in layer 5 and less frequently in layers 2-4 and 6 throughout neocortical regions. Combined labeling of immunofluorescence and fluorescent mRNA signals revealed that almost all PPD-immunopositive neurons expressed glutamic acid decarboxylase but not vesicular glutamate transporter, indicating their γ-aminobutyric acid (GABA)ergic characteristics, and that PPD-immunopositive neurons accounted for 15% of GABAergic interneurons in the primary somatosensory area. As GABAergic interneurons were divided into several groups by specific markers, we further examined the chemical characteristics of PPD-expressing neurons by the double immunofluorescence labeling method. More than 95% of PPD-immunopositive neurons were also somatostatin (SOM)-immunopositive in the primary somatosensory, primary motor, orbitofrontal, and primary visual areas, but only 24% were SOM-immunopositive in the medial prefrontal cortex. In the primary somatosensory area, PPD-immunopositive neurons constituted 50%, 79%, 55%, and 17% of SOM-immunopositive neurons in layers 2-3, 4, 5, and 6, respectively. Although SOM-expressing neurons contained calretinin-, neuropeptide Y-, nitric oxide synthase-, and reelin-expressing neurons as subgroups, only reelin immunoreactivity was detected in many PPD-immunopositive neurons. These results indicate that PPD-expressing neurons constitute a large subgroup of SOM-expressing cortical interneurons, and the PPD/SOM-expressing GABAergic neurons might serve not only as inhibitory elements in the local cortical circuit, but also as modulators for cortical neurons expressing κ-opioid and/or SOM receptors. Copyright © 2013 Wiley Periodicals, Inc.

Immunofluorescence-based methods to monitor DNA end resection

PubMed Central

Mukherjee, Bipasha; Tomimatsu, Nozomi; Burma, Sandeep

2017-01-01

Summary Double-strand breaks (DSBs) are the most deleterious amongst all types of DNA damage that can occur in the cell. These breaks arise from both endogenous (for example, DNA replication stress) as well as exogenous insults (for example, ionizing radiation). DSBs are principally repaired by one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error-prone process that can occur in all phases of the cell cycle, while HR is limited to the S and G2 phases of the cell cycle when a sister chromatid is available as a template for error-free repair. The first step in HR is “DNA end resection”, a process during which the broken DNA end is converted into a long stretch of 3′-ended single-stranded DNA (ssDNA). In recent years, DNA end resection has been identified as a pivotal step that controls “repair pathway choice” i.e., the appropriate choice between NHEJ and HR for DSB repair. Therefore, methods to quantitatively or semi-quantitatively assess DNA end resection have gained importance in laboratories working on DNA repair. In this chapter, we describe two simple immunofluorescence-based techniques to monitor DNA end resection in mammalian cells. The first technique involves immuno-detection of Replication Protein A (RPA), a ssDNA-binding protein that binds to resected DNA. The second technique involves labeling of genomic DNA with 5-bromo-2′-deoxyuridine (BrdU) that can be detected by anti-BrdU antibody only after the DNA becomes single stranded due to resection. These methods are not complicated, do not involve sophisticated instrumentation or reporter constructs, and can be applied to most mammalian cell lines, and therefore, should be of broad utility as simple ways of monitoring DNA end resection in vivo. PMID:25804748

Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex.

PubMed

Xi, Dong; Zhang, Wentong; Wang, Huai-Xing; Stradtman, George G; Gao, Wen-Jun

2009-11-01

N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

[Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies. Atypical images and their relationship with Chagas' disease and leishmaniasis].

PubMed

Griemberg, Gloria; Ferrarotti, Nidia F; Svibel, Graciela; Ravelli, Maria R; Taranto, Nestor J; Malchiodi, Emilio L; Pizzimenti, Maria C

2006-01-01

Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with Crithidia luciliae, displaying an annular image due to a kinetoplast containing double stranded DNA. Other structures such as membrane, flagellum and basal corpuscle can be stained as well, showing what is called atypical fluorescent images. As C. luciliae belongs to the Trypanosomatidae family, which include the human pathogens Trypanosoma cruzi and Leishmania spp., it was considered that these atypical images could be caused by cross-reactions. Serological studies for Chagas' disease were performed in 105 serum samples displaying atypical images. Sixty four percent of the samples from non endemic and 78.3% from endemic areas for Chagas' disease showed fluorescence in both, membrane and flagellum (joint image). Fifty samples from normal blood donors and 57 samples from patients with conective tissue diseases were tested with C. luciliae. None of them presented the joint image except for two patients with lupus who were also chagasic. In addition, 54 samples from chagasic patients were studied and all of them presented the joint image. We also studied 46 samples from patients with leishmaniasis from whom 28 were coinfected with T. cruzi. The joint image was observed in 88.0% of the samples with leishmaniasis and in 89.3% of the co-infected samples. The results suggest that C. luciliae could be used as an economical, and of low risk, alternative substrate for the serological diagnosis of Chagas' disease, even though it does not discriminate for Leishmania spp. infection. This study also suggests that whenever atypical images are observed in C. luciliae during the search for anti-DNA antibodies, it would be convenient to submit the patient to clinical and serological tests for the diagnosis of leishmaniosis and Chagas' disease.

Choline acetyltransferase-like immunofluorescence in epidermis of human skin.

PubMed

Johansson, O; Wang, L

1993-01-01

Using the indirect immunofluorescence approach the occurrence of choline acetyltransferase-like immunoreactivity in epidermis, except stratum basale, of human skin is described. Immunoreactive cells were also found in hair follicles, sweat gland ducts and sebaceous glands.

Application of information-theoretic measures to quantitative analysis of immunofluorescent microscope imaging.

PubMed

Shutin, Dmitriy; Zlobinskaya, Olga

2010-02-01

The goal of this contribution is to apply model-based information-theoretic measures to the quantification of relative differences between immunofluorescent signals. Several models for approximating the empirical fluorescence intensity distributions are considered, namely Gaussian, Gamma, Beta, and kernel densities. As a distance measure the Hellinger distance and the Kullback-Leibler divergence are considered. For the Gaussian, Gamma, and Beta models the closed-form expressions for evaluating the distance as a function of the model parameters are obtained. The advantages of the proposed quantification framework as compared to simple mean-based approaches are analyzed with numerical simulations. Two biological experiments are also considered. The first is the functional analysis of the p8 subunit of the TFIIH complex responsible for a rare hereditary multi-system disorder--trichothiodystrophy group A (TTD-A). In the second experiment the proposed methods are applied to assess the UV-induced DNA lesion repair rate. A good agreement between our in vivo results and those obtained with an alternative in vitro measurement is established. We believe that the computational simplicity and the effectiveness of the proposed quantification procedure will make it very attractive for different analysis tasks in functional proteomics, as well as in high-content screening. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Immunofluorescence detection of pea protein in meat products.

PubMed

Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

2016-08-01

In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

USAFSAM (USAF School of Aerospace Medicine) Review and Analysis of Radiofrequency Radiation Bioeffects Literature: Fourth Report.

DTIC Science & Technology

1984-05-01

TEST CHART ?NATIONAL BUREAU OF STANDARDS-1963-A AD)A142 961 Repor USAFSAM-TR-84-17 USAFSAM REVIEW AND ANALYSIS OF RADIOFREQUENCY RADIATION BIOEFFECTS...1983a) AUTHOR ABSTRACT: Normal mouse B lymphocytes were tested for the ability to cap plasma antigen-antibody complexes following exposure to 2.45-GHz...treatment, the irradiated cells and the nonirradiated controls were tested for capping by the direct immunofluorescence technique. First, the cells

Peroxisome proliferator-activated receptor (PPAR)gamma is highly expressed in normal human pituitary gland.

PubMed

Bogazzi, F; Russo, D; Locci, M T; Chifenti, B; Ultimieri, F; Raggi, F; Viacava, P; Cecchetti, D; Cosci, C; Sardella, C; Acerbi, G; Gasperi, M; Martino, E

2005-11-01

Expression of peroxisome proliferator-activated receptor (PPAR)gamma in normal pituitary seems to be restricted to ACTH-secreting cells. The aim of the study was to evaluate the expression of PPARgamma in normal human pituitary tissue and to study its localization in the pituitary secreting cells. Normal pituitary tissue samples were obtained form 11 patients with non-secreting adenoma who underwent surgical excision of the tumor. Expression of PPARgamma was evaluated by immunostaining and western blotting; localization of PPARgamma in each pituitary secreting cell lineage was evaluated by double immunofluorescence using confocal microscopy. Pituitary non-functioning adenomas served as Controls. PPARgamma was highly expressed in all pituitary samples with a (mean +/- SD) 81 +/- 6.5% of stained cells; expression of PPARgamma was confirmed by western blotting. Non-functioning pituitary adenomas had 74 +/- 11% PPARgamma positive cells. Expression of PPARy was either in cytoplasm or nuclei. In addition, treatment of GH3 cells, with a PPARgamma ligand was associated with traslocation of the receptor from cytoplasm into the nucleus. Double immunostaining revealed that every pituitary secreting cell (GH, TSH, LH, FSH, PRL and ACTH) had PPARgamma expressed. The present study demonstrated that PPARgamma is highly expressed in every normal pituitary secreting cell lineage. It can translocate into the nucleus by ligand binding; however, its role in pituitary hormone regulation remains to be elucidated.

Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

PubMed Central

Castro-Pérez, Edgardo; Soto-Soto, Emilio; Pérez-Carambot, Marizabeth; Dionisio-Santos, Dawling; Saied-Santiago, Kristian; Ortiz-Zuazaga, Humberto G.; Peña de Ortiz, Sandra

2016-01-01

An increasing body of evidence suggests that mechanisms related to the introduction and repair of DNA double strand breaks (DSBs) may be associated with long-term memory (LTM) processes. Previous studies from our group suggested that factors known to function in DNA recombination/repair machineries, such as DNA ligases, polymerases, and DNA endonucleases, play a role in LTM. Here we report data using C57BL/6 mice showing that the V(D)J recombination-activating gene 1 (RAG1), which encodes a factor that introduces DSBs in immunoglobulin and T-cell receptor genes, is induced in the amygdala, but not in the hippocampus, after context fear conditioning. Amygdalar induction of RAG1 mRNA, measured by real-time PCR, was not observed in context-only or shock-only controls, suggesting that the context fear conditioning response is related to associative learning processes. Furthermore, double immunofluorescence studies demonstrated the neuronal localization of RAG1 protein in amygdalar sections prepared after perfusion and fixation. In functional studies, intra-amygdalar injections of RAG1 gapmer antisense oligonucleotides, given 1 h prior to conditioning, resulted in amygdalar knockdown of RAG1 mRNA and a significant impairment in LTM, tested 24 h after training. Overall, these findings suggest that the V(D)J recombination-activating gene 1, RAG1, may play a role in LTM consolidation. PMID:26843989

Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells.

PubMed

Krishnamurthy, Nirmala; Liu, Lili; Xiong, Xiahui; Zhang, Junran; Montano, Monica M

2015-01-01

Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.

Structural Configuration Systems Analysis for Advanced Aircraft Fuselage Concepts

NASA Technical Reports Server (NTRS)

Mukhopadhyay, Vivek; Welstead, Jason R.; Quinlan, Jesse R.; Guynn, Mark D.

2016-01-01

Structural configuration analysis of an advanced aircraft fuselage concept is investigated. This concept is characterized by a double-bubble section fuselage with rear mounted engines. Based on lessons learned from structural systems analysis of unconventional aircraft, high-fidelity finite-element models (FEM) are developed for evaluating structural performance of three double-bubble section configurations. Structural sizing and stress analysis are applied for design improvement and weight reduction. Among the three double-bubble configurations, the double-D cross-section fuselage design was found to have a relatively lower structural weight. The structural FEM weights of these three double-bubble fuselage section concepts are also compared with several cylindrical fuselage models. Since these fuselage concepts are different in size, shape and material, the fuselage structural FEM weights are normalized by the corresponding passenger floor area for a relative comparison. This structural systems analysis indicates that an advanced composite double-D section fuselage may have a relative structural weight ratio advantage over a conventional aluminum fuselage. Ten commercial and conceptual aircraft fuselage structural weight estimates, which are empirically derived from the corresponding maximum takeoff gross weight, are also presented and compared with the FEM- based estimates for possible correlation. A conceptual full vehicle FEM model with a double-D fuselage is also developed for preliminary structural analysis and weight estimation.

Molecular Histopathology Using Gold Nanorods and Optical Coherence Tomography

PubMed Central

Prabhulkar, Shradha; Matthews, Jared; Rawal, Siddarth; Awdeh, Richard M.

2013-01-01

Purpose. To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). Methods. GNRs were synthesized and covalently attached to anti–glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti–GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. Results. No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. Conclusions. We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal. PMID:23307958

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

Correlation between serological and immunofluorescence results in the investigation of brucellosis in veterinary surgeons.

PubMed Central

Henderson, R J; Hill, D M; Vickers, A A; Edwards, J M; Tillett, H

1976-01-01

Four serological tests and three immunofluorescence tests for IgG, IgM, and IgA were compared for value in the investigation of brucellosis in veterinary surgeons. No one serological test stood out over the others, and the immunofluorescence tests did not appear to have advantages over the serological tests. If a laboratory is limited in time and resources then the saline agglutination or the complement fixation test would be reasonably satisfactory. The 2-mercaptoethanol test and the antihuman globulin (Coombs' test) have no advantages over the other two and could be dropped. Immunofluorescence tests are not recommended for routine testing of brucellosis sera. The results and these recommendations apply to the 'vet' sera tested; it is reasonable to suppose that what applies to 'vet' sera will also apply to sera of those who work with or are in repeated contact with cattle and who will have had previous experience of brucella antigen, that is, dairy farmers, herdsmen, or slaughter house employees. PMID:765361

[Establishment of systemic lupus erythematosus-like murine model with Sm mimotope].

PubMed

Xie, Hong-Fu; Feng, Hao; Zeng, Hai-Yan; Li, Ji; Shi, Wei; Yi, Mei; Wu, Bin

2007-04-01

To establish systemic lupus erythematosus (SLE) -like murine model by immunizing BALB/C mice with Sm mimotope. Sm mimotope was identified by screening a 12-mer random peptide library with monoclonal anti-Smith antibody. Sm mimotope was initially defined with sandwich ELISA, DNA sequencing, and deduced amino acid sequence; and BALB/C mice were subcutaneously injected with mixture phages clones. Sera Sm antibody, anti-double stranded DNA (dsDNA) antibody, and antinuclear antibody (ANA) of mice were detected using direct immunofluorescence; kidney histological changes were examined by HE staining. Five randomly selected peptides were sequenced and the amino acid sequences IR, SQ, and PP were detected in a higher frequency. High-titer IgG autoantibodies of dsDNA, Sm, and ANA in the sera of experiment group were detected by ELISA 28 days after having been immunized by Sm mimotope. Proteinuria was detected 33 days later; immune complex and nephritis were observed in kidney specimens. SLE-like murine model can be successfully induced by Sm phage mimotope.

Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology.

PubMed

Ngolab, Jennifer; Trinh, Ivy; Rockenstein, Edward; Mante, Michael; Florio, Jazmin; Trejo, Margarita; Masliah, Deborah; Adame, Anthony; Masliah, Eliezer; Rissman, Robert A

2017-06-09

Proteins implicated in neurodegenerative conditions such as Alzheimer's disease (AD) and Dementia with Lewy Bodies (DLB) have been identified in bodily fluids encased in extracellular vesicles called exosomes. Whether exosomes found in DLB patients can transmit pathology is not clear. In this study, exosomes were successfully harvested through ultracentrifugation from brain tissue from DLB and AD patients as well as non-diseased brain tissue. Exosomes extracted from brains diagnosed with either AD or DLB contained aggregate-prone proteins. Furthermore, injection of brain-derived exosomes from DLB patients into the brains of wild type mice induced α-synuclein (α-syn) aggregation. As assessed through immunofluorescent double labeling, α-syn aggregation was observed in MAP2 + , Rab5 + neurons. Using a neuronal cell line, we also identified intracellular α-syn aggregation mediated by exosomes is dependent on recipient cell endocytosis. Together, these data suggest that exosomes from DLB patients are sufficient for seeding and propagating α-syn aggregation in vivo.

Nitric Oxide Signaling in Hypergravity-Induced Neuronal Plasticity

NASA Technical Reports Server (NTRS)

Holstein, Gay R.

2003-01-01

The goal of this research project was to identify the neurons and circuits in the vestibular nuclei and nucleus prepositus hypoglossi that utilize nitric oxide (NO) for intercellular signaling during gravity-induced plasticity. This objective was pursued using histochemical and immunocytochemical approaches to localize NO-producing neurons and characterize the fine morphology of the cells in ground-based studies of normal rats, rats adapted to hypergravity, and rats adapted to hypergravity and then re-adapted to the 1G environment. NO-producing neurons were identified and studied using four methodologies: i) immunocytochemistry employing polyclonal antibodies directed against neuronal nitric oxide synthase (nNOS), to provide an indication of the capacity of a cell for NO production; ii) immunocytochemistry employing a monoclonal antibody directed against L-citrulline, to provide an indirect index of the enzyme's activity; iii) histochemistry based on the NADPH-diaphorase reaction, for fuI1 cytological visualization of neurons; and iv) double immunofluorescence to co-localize nNOS and L-citrulline in individual vestibular nuclei (VN) and neurons.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis.

PubMed

Wang, Y T; Liu, W; Seah, J N; Lam, C S; Xiang, J H; Korzh, V; Kwang, J

2002-12-10

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

PubMed Central

Barrasa, J. M.; Gutiérrez, A.; Escaso, V.; Guillén, F.; Martínez, M. J.; Martínez, A. T.

1998-01-01

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw. PMID:9435085

Bovine papillomavirus type 2 infects the urinary bladder of water buffalo (Bubalus bubalis) and plays a crucial role in bubaline urothelial carcinogenesis.

PubMed

Roperto, Sante; Russo, Valeria; Ozkul, Ayhan; Sepici-Dincel, Aylin; Maiolino, Paola; Borzacchiello, Giuseppe; Marcus, Ioan; Esposito, Iolanda; Riccardi, Marita Georgia; Roperto, Franco

2013-02-01

Bovine papillomavirus type 2 (BPV-2) has been shown to infect and play a role in urinary bladder carcinogenesis of buffaloes grazed on pastures with ferns from the Marmara and Black Sea Regions of Turkey. BPV-2 DNA has been found in both neoplastic and non-neoplastic lesions of the urinary bladder. Furthermore, this virus may be a normal inhabitant of the urinary bladder since BPV-2 DNA has also been detected in clinically normal buffaloes. The viral activation by fern immunosuppressant or carcinogen may trigger the urothelial cell transformation. The E5 oncoprotein was solely detected in urothelial tumours and appeared to be co-localized with the overexpressed and phosphorylated platelet derived growth factor (PDGF) β receptor in a double-colour immunofluorescence assay. Our results indicate that the E5-PDGF β receptor interaction also occurs in spontaneous tumours of the bubaline urinary bladder, revealing an additional role of BPV-2 in bladder carcinogenesis of buffaloes.

Systemic lupus erythematosus and renal tubular acidosis associated with hyperthyroidism. Case Report.

PubMed

Deng, Datong; Sun, Li; Xia, Tongjia; Xu, Min; Wang, Youmin; Zhang, Qiu

2016-07-01

A case of a 42-year-old female with hyperthyroidism was subsequently diagnosed to have systemic lupus erythematosus with distal RTA. The clinical examination on admission showed swelling of the knee joints and the urinalysis showed pH 6.5, pro 3+. Her blood routine results were as follows: white blood cells 1.85×109/L, platelets 100×109/L, erythrocyte 3.06×1012/L. The serum potassium was 3.11 mmol/L, 24 hour urinary electrolyte: K 68.87 mmol/24 H, antinuclear antibodies (ANA) 1:1 000, speckled pattern. The anti-double stranded DNA antibody (anti-dsDNA), anti SS-A(52) antibody and anti SS-A(60) antibody were positive. The light microscopy and immunofluorescence showed diffuse proliferative lupus nephritis. These data were compatible with the diagnosis of systemic lupus erythematosus. The diagnosis of hyperthyroidism and distal RTA is clear. This report showed that other autoimmune disease in the diagnosis of hyperthyroidism should not be ignored.

Immunohistochemical Localization of Periostin in Human Gingiva

PubMed Central

Cobo, T.; Obaya, A.; Cal, S.; Solares, L.; Cabo, R.; Vega, J.A.; Cobo, J.

2015-01-01

The periostin is a matricellular protein expressed in collagen-rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces, growth factors and cytokines. Interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated. Here we used Western blot and double immunofluorescence coupled to laser-confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects. By Western blot a protein band with an estimated molecular mass of 94 kDa was observed. Periostin was localized at the epithelial-connective tissue junction, or among the fibers of the periodontal ligament, and never co-localized with cytokeratin or vimentin thus suggesting it is an extracellular protein. These results demonstrate the occurrence of periostin in adult human gingiva; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells, and therefore in the physiopathological conditions in which these interactions are altered. PMID:26428890

Association of plasma cell subsets in the bone marrow and free light chain concentrations in the serum of monoclonal gammopathy patients.

PubMed

Ayliffe, Michael John; Behrens, Judith; Stern, Simon; Sumar, Nazira

2012-08-01

This study investigated bone marrow plasma cell subsets and monoclonal free light chain concentrations in blood of monoclonal gammopathy patients. 54 bone marrow samples were stained by double immunofluorescence to enumerate cellular subsets making either intact monoclonal immunoglobulin or free light chains only. Blood taken at the same time was assayed for free light chains by an automated immunoassay. Patients were assigned to three cellular population categories: single intact monoclonal immunoglobulin (59%), dual monoclonal immunoglobulin and free light chain only (31%), or single free light chain only (9%). The median affected free light chain concentration of each group was 75 mg/l, 903 mg/l and 3320 mg/l, respectively, but with substantial overlap. In myeloma patients the difference in serum free light chain concentrations between patients with free light chain only marrow cells and those without was statistically significant. Serum free light chain levels >600 mg/l result mostly from marrow cells restricted to free light chain production.

BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

EPA Science Inventory

An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

PubMed

Erlandsen, S L; Sherlock, L A; Bemrick, W J

1990-04-01

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

PubMed

Dobrzynski, H; Marples, D D; Musa, H; Yamanushi, T T; Henderson, Z; Takagishi, Y; Honjo, H; Kodama, I; Boyett, M R

2001-10-01

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis

PubMed Central

Mahavadi, Poornima; Sasikumar, Satish; Cushing, Leah; Hyland, Tessa; Rosser, Ann E.; Riccardi, Daniela; Lu, Jining; Kalin, Tanya V.; Kalinichenko, Vladimir V.; Guenther, Andreas; Ramirez, Maria I.; Pardo, Annie; Selman, Moisés; Warburton, David

2013-01-01

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor, maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar mesenchymal reexpression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial vs. mesenchymal markers. Grhl2 was not detected in mesenchyme in intraperitoneal bleomycin-induced injury as well as in spontaneously occurring fibrosis in double-mutant HPS1 and HPS2 mice, whereas in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further validated by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important pathophysiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity. PMID:24375798

Localization and signaling patterns of vascular endothelial growth factors and receptors in human periapical lesions.

PubMed

Virtej, Anca; Løes, Sigbjørn S; Berggreen, Ellen; Bletsa, Athanasia

2013-05-01

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions. Tissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples. The histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated. The cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Establishment and characterization of a novel osteosarcoma cell line: CHOS.

PubMed

Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

2016-12-01

Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Expression of sigma receptor 1 mRNA and protein in rat retina.

PubMed

Liu, L L; Wang, L; Zhong, Y M; Yang, X L

2010-06-02

Sigma receptor (sigmaR), known as a unique nonopiate, nonphencyclidine brain receptor, can bind diverse classes of psychotropic drugs, neurosteroids and other synthetic compounds, such as (+)pentazocine, etc. Two types of sigmaRs have been identified: sigmaR1 and sigmaR2. In this work, we examined the expression of sigmaR1 in rat retina by reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling. RT-PCR analysis showed that sigmaR1 mRNA was present in rat retina. Furthermore, labeling for sigmaR1 was diffusely distributed in the outer and inner plexiform layers. The sigmaR1-immunoreactivity (IR) was also observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina sigmaR1 was expressed in all horizontal cells labeled by calbindin. In contrast, no sigmaR1-IR was detected in several subtypes of bipolar cells, including rod-dominant ON-type bipolar cells, types 2, 3, 5 and 8 bipolar cells, labeled by protein kinase C (PKC), recoverin and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) respectively. In the inner retina, most of GABAergic amacrine cells, including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, expressed sigmaR1. Some glycinergic amacrine cells were also labeled by sigmaR1, but glycinergic AII amacrine cells were not labeled. In addition, sigmaR1-IR was seen in almost all somata of the ganglion cells retrogradely labeled by fluorogold. These results suggest that sigmaR1 may have neuromodulatory and neuroprotective roles in the retina. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

PubMed Central

Niikura, Yohei; Kitagawa, Katsumi

2016-01-01

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells. PMID:26967065

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

PubMed

Fujimaki-Aoba, Kayo; Tanaka, Kayoko; Inomata, Reiko; Jensik, Philip J; Takada, Makoto

2014-01-01

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

PubMed Central

Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Jaramillo, C; Castañon, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Warhurst, D C

1992-01-01

Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Images PMID:1452651

Expression and analysis of exogenous proteins in epidermal cells.

PubMed

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

Lineage Analysis in Pulmonary Arterial Hypertension

DTIC Science & Technology

2013-06-01

monocrotaline pyrrole . The fate of lacZ-expressing cells will be correlated with immunofluorescent staining of endothelial marker CD31, mesenchymal marker...A) Pilot study: Time course of development of pulmonary hypertension in pneumonectomized mice injected with monocrotaline pyrrole (P/MCTP, n = 4... pyrrole . A, B) Normal muscular pulmonary artery (PA) adjacent to bronchiole (Br) A) hematoxylin and eosin stain (H&E), B) elastin-van Gieson stain (EVG

Familial primary cutaneous amyloidosis. Clinical, genetic, and immunofluorescent studies.

PubMed

Vasily, D B; Bhatia, S G; Uhlin, S R

1978-08-01

Familial primary cutaneous amyloidosis, a rare, autosomal dominant genodermatosis, affected 16 of 46 family members of German descent. Previous case reports involved families of Russian, Spanish, or Chinese descent. The finding of IgG, IgM, C3 in the amyloid deposits confirms recent reports of immunofluorescent dermal amyloid deposits.

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.

Establishment and characterization of pygmy killer whale (Feresa attenuata) dermal fibroblast cell line.

PubMed

Yajing, Sun; Rajput, Imran Rashid; Ying, Huang; Fei, Yu; Sanganyado, Edmond; Ping, Li; Jingzhen, Wang; Wenhua, Liu

2018-01-01

The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham's F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW dermal fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes.

PubMed

Bader, Hannah L; Keene, Douglas R; Charvet, Benjamin; Veit, Guido; Driever, Wolfgang; Koch, Manuel; Ruggiero, Florence

2009-01-01

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII alpha1 chain was characterized as a collagenase sensitive band migrating at approximately 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.

Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement

PubMed Central

Zarrella, Elizabeth; Coulter, Madeline; Welsh, Allison; Carvajal, Daniel; Schalper, Kurt; Harigopal, Malini; Rimm, David; Neumeister, Veronique

2016-01-01

While FDA approved methods of assessment of Estrogen Receptor (ER) are “fit for purpose”, they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperio’s nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the AQUA® method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R2 > 0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan-Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026), but there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative, in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps due to low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests. PMID:27348626

Analysis and Design of a Double-Divert Spiral Groove Seal

NASA Technical Reports Server (NTRS)

Zheng, Xiaoqing; Berard, Gerald

2007-01-01

This viewgraph presentation describes the design and analysis of a double spiral groove seal. The contents include: 1) Double Spiral Design Features; 2) Double Spiral Operational Features; 3) Mating Ring/Rotor Assembly; 4) Seal Ring Assembly; 5) Insert Segment Joints; 6) Rotor Assembly Completed Prototype Parts; 7) Seal Assembly Completed Prototype Parts; 8) Finite Element Analysis; 9) Computational Fluid Dynamics (CFD) Analysis; 10) Restrictive Orifice Design; 11) Orifice CFD Model; 12) Orifice Results; 13) Restrictive Orifice; 14) Seal Face Coning; 15) Permanent Magnet Analysis; 16) Magnetic Repulsive Force; 17) Magnetic Repulsive Test Results; 18) Spin Testing; and 19) Testing and Validation.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

PubMed Central

Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

2016-01-01

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

HEXIM1 is a promiscuous double-stranded RNA-binding protein and interacts with RNAs in addition to 7SK in cultured cells

PubMed Central

Li, Qintong; Cooper, Jeffrey J.; Altwerger, Gary H.; Feldkamp, Michael D.; Shea, Madeline A.; Price, David H.

2007-01-01

P-TEFb regulates eukaryotic gene expression at the level of transcription elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein HEXIM1 or HEXIM2. In an effort to determine the minimal region of 7SK needed to interact with HEXIM1 in vitro, we found that an oligo comprised of nucleotides 10–48 sufficed. A bid to further narrow down the minimal region of 7SK led to a surprising finding that HEXIM1 binds to double-stranded RNA in a sequence-independent manner. Both dsRNA and 7SK (10–48), but not dsDNA, competed efficiently with full-length 7SK for HEXIM1 binding in vitro. Upon binding dsRNA, a large conformational change was observed in HEXIM1 that allowed the recruitment and inhibition of P-TEFb. Both subcellular fractionation and immunofluorescence demonstrated that, while most HEXIM1 is found in the nucleus, a significant fraction is found in the cytoplasm. Immunoprecipitation experiments demonstrated that both nuclear and cytoplasmic HEXIM1 is associated with RNA. Interestingly, the one microRNA examined (mir-16) was found in HEXIM1 immunoprecipitates, while the small nuclear RNAs, U6 and U2, were not. Our study illuminates novel properties of HEXIM1 both in vitro and in vivo, and suggests that HEXIM1 may be involved in other nuclear and cytoplasmic processes besides controlling P-TEFb. PMID:17395637

Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

PubMed

Shirol, Pallavi D; Naik, Veena; Kale, Alka

2015-10-01

Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

Comparison of three methods for the detection of Trichinella spiralis infections in pigs by five European laboratories*

PubMed Central

Kohler, G.; Ruitenberg, E. J.

1974-01-01

Three methods employed in the diagnosis of trichinosis (trichinoscopy, digestion method, and immunofluorescence technique) were compared by laboratories in 5 countries of the European economic community. For this purpose, material from 32 pigs infected with 50, 150, 500, and 1 500 T. spiralis larvae was examined. With none of the three methods was it possible to detect with sufficient reliability a T. spiralis infection in pigs infected with 50 larvae. The digestion method and the immunofluorescence technique yielded more reliable results when the infection dose was 150 larvae or more. With trichinoscopy, reliable results were obtained in pigs infected with 500 and 1 500 larvae. With the digestion method and trichinoscopy, the onset of infections was detectable from 3 weeks post infection, the digestion method being more reliable; the immunofluorescence technique yielded positive results from approximately 4-6 weeks post infection. The immunofluorescence technique is applicable for epidemiological surveys. As a routine diagnostic procedure in the slaughterhouse, trichinoscopy and the digestion method are possible alternatives, the latter being more sensitive. PMID:4616776

Autoantibodies against Leydig cells in patients after spermatic cord torsion.

PubMed Central

Zanchetta, R; Mastrogiacomo, I; Graziotti, P; Foresta, C; Betterle, C

1984-01-01

This study is aimed at searching for the presence of circulating antibodies against frozen sections of human testis, ovary and trophoblast in patients that had spermatic cord torsion. Sixty-eight sera samples were studied. Nine patients (13.2%) were positive for organ specific anti-testis autoantibodies. Six patients were positive for antibodies against Leydig cells: five were positive only with the indirect immunofluorescence technique of complement fixing (ITT/CF), the sixth patient was positive only with the indirect immunofluorescence technique (ITT). The other three patients were positive for antibodies against germ line cells: two patients were positive with both techniques, the third was positive only with indirect immunofluorescence technique. Eight of these patients were negative for antibodies against adrenal cortex while only one case was positive with indirect immunofluorescence technique both on adrenal cortex and Leydig cells. Human lyophilized testis absorbed the reactive antibodies against Leydig cells and germ line cells, while adrenal cortex and lyophilized testosterone were ineffective. This study shows the identification of a specific antibody against Leydig cells and germ line cells in patients after spermatic cord torsion. PMID:6362937

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool.

PubMed

Mesquita, Flávio da Silva; Oliveira, Danielle Bruna Leal de; Crema, Daniela; Pinez, Célia Miranda Nunes; Colmanetti, Thaís Cristina; Thomazelli, Luciano Matsumia; Gilio, Alfredo Elias; Vieira, Sandra Elisabeth; Martinez, Marina Baquerizo; Botosso, Viviane Fongaro; Durigon, Edison Luiz

The aim of this study was to evaluate the QuickVue ® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue ® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue ® RSV Test and viral load or specific strain. The QuickVue ® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. This study demonstrated that the QuickVue ® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

A topical aqueous oxygen emulsion stimulates granulation tissue formation in a porcine second-degree burn wound.

PubMed

Li, Jie; Zhang, Yan-Ping; Zarei, Mina; Zhu, Linjian; Sierra, Jose Ollague; Mertz, Patricia M; Davis, Stephen C

2015-08-01

Oxygen is an essential substance for wound healing. Limited studies have shown that topical oxygen can influence healing. This study evaluated the effects of a Topical Oxygen Emulsion (TOE) on burn wound healing. A porcine second-degree burn wound model was used in the study. Burn wounds were randomly assigned to TOE, vehicle control, and no-treatment (air) groups. Effects of TOE on the granulation tissue formation and angiogenesis were studied using hematoxylin and eosin histological analysis. Protein production and gene expression of types I and III collagen and vascular endothelial growth factor (VEGF) were determined using immunofluorescent staining and Reverse Transcription and Polymerase Chain Reaction (RT-PCR), respectively. The TOE treated wounds exhibited better angiogenesis and granulation tissue formation by histology examination. The immunofluorescence staining and RT-PCR analysis demonstrated that protein production and mRNA expression of VEGF and collagen III were significantly higher in TOE treatment group than vehicle alone and air control groups, while there was no significant difference in the level of collagen I. Our data demonstrate that TOE enhances burn wound healing via stimulating the expression of VEGF and type III collagen and strongly indicates the potential use of TOE in wounds. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

VPS53 mutations cause progressive cerebello-cerebral atrophy type 2 (PCCA2).

PubMed

Feinstein, Miora; Flusser, Hagit; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Lev, Dorit; Agamy, Orly; Cohen, Idan; Kadir, Rotem; Sivan, Sara; Leshinsky-Silver, Esther; Markus, Barak; Birk, Ohad S

2014-05-01

Progressive cerebello-cerebral atrophy (PCCA) leading to profound mental retardation, progressive microcephaly, spasticity and early onset epilepsy, was diagnosed in four non-consanguineous apparently unrelated families of Jewish Moroccan ancestry. Common founder mutation(s) were assumed. Genome-wide linkage analysis and whole exome sequencing were done, followed by realtime PCR and immunofluorescent microscopy. Genome-wide linkage analysis mapped the disease-associated gene to 0.5 Mb on chromosome 17p13.3. Whole exome sequencing identified only two mutations within this locus, which were common to the affected individuals: compound heterozygous mutations in VPS53, segregating as expected for autosomal recessive heredity within all four families, and common in Moroccan Jews (∼1:37 carrier rate). The Golgi-associated retrograde protein (GARP) complex is involved in the retrograde pathway recycling endocytic vesicles to Golgi; c.2084A>G and c.1556+5G>A VPS53 founder mutations are predicted to affect the C-terminal domain of VPS53, known to be critical to its role as part of this complex. Immunofluorescent microscopy demonstrated swollen and abnormally numerous CD63 positive vesicular bodies, likely intermediate recycling/late endosomes, in fibroblasts of affected individuals. Autosomal recessive PCCA type 2 is caused by VPS53 mutations.

Livedoid vasculopathy in a patient with factor V mutation (Leiden).

PubMed

Biedermann, T; Flaig, M J; Sander, C A

2000-09-01

Frequently, no underlying disease can be detected in patients with livedoid vasculopathy. For these forms, an unknown vaso-occlusive or thrombogenic process has been accused to play a role. Thus, a patient with livedoid vasculopathy was examined for different parameters which can be involved in coagulopathies. Laboratory studies for different autoantigen reactive immunoglobulins, cryoglobulins, and circulating immune complexes were carried out. Besides dermatopathologic examination, a biopsy specimen was analyzed by direct immunofluorescence for immunoglobulin (Ig) and complement deposits. Furthermore, hemostaseological function tests including activated protein C (APC) resistance were undertaken. Positive only at very low titres were antinuclear antibodies and c-ANCA, all other parameters were within normal ranges or negative. Direct immunofluorescence revealed IgM, C3 and fibrogen deposits. Hemostaseological function tests demonstrated a pathologic activated protein c resistance and PCR analysis a heterozygous defect of the factor V (Leiden). The diagnosis of livedoid vasculopathy associated with factor V mutation (Leiden) was made. Since the underlying cause for livedoid vasculopathy often remains unknown, we suggest that hemostaseological function tests including APC resistance and factor V gene mutation analysis should be carried out. Further studies have to follow in order to elucidate the role of mutant factor V in livedoid vasculopathy and in cutaneous ulcerations.

The intrinsic stiffness of human trabecular meshwork cells increases with senescence

PubMed Central

Chang, Yow-Ren; Murphy, Christopher J.; Russell, Paul

2015-01-01

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases. PMID:25915531

The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast.

PubMed

Rajvanshi, Praveen Kumar; Arya, Madhuri; Rajasekharan, Ram

2017-11-10

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2 Δ msn4 Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2 Δ msn4 Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2 Δ msn4 Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2 Δ msn4 Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2 Δ msn4 Δ strain could therefore be a good model system for further study of these critical processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellulose/soy protein composite-based nerve guidance conduits with designed microstructure for peripheral nerve regeneration

NASA Astrophysics Data System (ADS)

Gan, Li; Zhao, Lei; Zhao, Yanteng; Li, Ke; Tong, Zan; Yi, Li; Wang, Xiong; Li, Yinping; Tian, Weiqun; He, Xiaohua; Zhao, Min; Li, Yan; Chen, Yun

2016-10-01

Objective. The objective of this work was to develop nerve guidance conduits from natural polymers, cellulose and soy protein isolate (SPI), by evaluating the effects of cellulose/SPI film-based conduit (CSFC) and cellulose/SPI sponge-based conduit (CSSC) on regeneration of nerve defects in rats. Approach. CSFC and CSSC with the same chemical components were fabricated from cellulose and SPI. Effects of CSSC and CSFC on regeneration of the defective nerve were comparatively investigated in rats with a 10 mm long gap in sciatic nerve. The outcomes of peripheral nerve repair were evaluated by a combination of electrophysiological assessment, Fluoro-Gold retrograde tracing, double NF200/S100 immunofluorescence analysis, toluidine blue staining, and electron microscopy. The probable molecular mechanism was investigated using quantitative real-time PCR (qPCR) analysis. Main results. Compared with CSFC, CSSC had 2.69 times higher porosity and 5.07 times higher water absorption, thus ensuring much higher permeability. The nerve defects were successfully bridged and repaired by CSSC and CSFC. Three months after surgery, the CSSC group had a higher compound muscle action potential amplitude ratio, a higher percentage of positive NF200 and S100 staining, and a higher axon diameter and myelin sheath thickness than the CSFC group, showing the repair efficiency of CSSC was higher than that of CSFC. qPCR analysis indicated the mRNA levels of nerve growth factor, IL-10, IL-6, and growth-associated protein 43 (GAP-43) were higher in the CSSC group. This also indicated that there was better nerve repair with CSSC due to the higher porosity and permeability of CSSC providing a more favourable microenvironment for nerve regeneration than CSFC. Significance. A promising nerve guidance conduit was developed from cellulose/SPI sponge that showed potential for application in the repair of nerve defect. This work also suggests that nerve guidance conduits with better repair efficiency could be developed through structure design and processing optimization.

Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment.

PubMed

Raghuveer, K; Senthilkumaran, B

2009-05-01

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

[Efficacy of combination therapy with pegylated-interferon alfa-2a plus ribavirin in autoantibody-positive chronic hepatitis C patients].

PubMed

Li, Ya-xin; Yang, Yan-jia; Yang, Mei; Chen, Li-yu; Lu, Jia-jie; Ma, Yuan-ji; Liu, Kai; Lei, Xue-zhong; Tang, Hong

2013-05-01

To evaluate the therapeutic efficacy of antiviral combination therapy with pegylated-interferon alpha-2a plus ribavirin (RBV) in patients with autoantibody-positive chronic hepatitis C (CHC) and to investigate the impact of the presence of autoantibodies on the treatment outcome. Eighty-six consecutive CHC patients who underwent a 48-week treatment regimen composed of Peg-IFNa-2a (135 or 180 mug/wk) plus weight-based RBV ( less than or equal to 65 kg, 800 mg/d; 65 to 75 kg, 1000 mg/d; more than or equal to75 kg, 1200 mg/d ). Prior to treatment (baseline) and at end of treatment (EOT; week 48), levels of antinuclear antibody (ANA), anti-smooth muscle antibody (SMA), anti liver/kidney microsomal antibody type 1 (LKM1), anti-La (SSB), and anti liver cytosolic-1 (LC-1) were detected by indirect immunofluorescence. At baseline, during treatment (weeks 4, 12, 24, and 36), EOT, and 24 weeks after EOT, levels of HCV RNA were assessed by real-time quantitative PCR. Rapid virological response (RVR) was defined as HCV RNA less than 10(3) copy/ml at week 4. Sustained virologic response (SVR) was defined as HCV RNA load below the lower limit of detection at 24 weeks after EOT. Correlation between autoantibodies and treatment-induced reduced HCV RNA load was assessed by univariate analysis of variance or chi-squared tests. Autoantibodies were detected in 24 patients, which included 14 ANA-positive patients, five SMA-positive patients, three LKM1-positive patients, one patient with double-positivity for ANA and SSB, and one patient with double-positivity for ANA and LC-1. The autoantibody-positive patients and autoantibody-negative patients showed similar rates of RVR (70.8% vs. 72.5%, P more than 0.05) and SVR (81.4% vs. 82.2%, P more than 0.05). Antiviral therapy with Peg-IFNa-2a RBV can effectively reduce the HCV RNA load in autoantibody-positive CHC patients; however, the presence of autoantibodies may not be an independent predictor of therapy outcome.

Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Fuseler, John W.; Merrill, Dana M.; Rogers, Jennifer A.; Grisham, Matthew B.; Wolf, Robert E.

2006-07-01

Nuclear factor kappa B (NF-[kappa]B) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-[kappa]Bs, which prevent entry into the nucleus. Following cellular stimulation, the I-[kappa]Bs are rapidly degraded, activating NF-[kappa]B. The active form of NF-[kappa]B rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-[kappa]B is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-[kappa]B (p65 subunit, p65-NF-[kappa]B) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-[kappa]B protein in the nucleus determined biochemically. The appearance of p65-NF-[kappa]B in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-[kappa]B can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

Protein regulation of induced pluripotent stem cells by transplanting in a Huntington's animal model.

PubMed

Mu, S; Han, L; Zhou, G; Mo, C; Duan, J; He, Z; Wang, Z; Ren, L; Zhang, J

2016-10-01

The purpose of this study was to determine the functional recovery and protein regulation by transplanted induced pluripotent stem cells in a rat model of Huntington's disease (HD). In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle 10 days after the quinolinic acid injection. At 8 weeks after transplantation, fluorodeoxyglucose-PET/CT scan and balance-beam test were performed to evaluate the functional recovery of experimental rats. In addition, immunofluorescence and protein array analysis were used to investigate the regulation of stimulated protein expression in the striatum. At 8 weeks after induced pluripotent stem cell transplantation, motor function was improved in comparison with the quinolinic acid-treated rats. High fluorodeoxyglucose accumulation in the injured striatum was also observed by PET/CT scans. In addition, immunofluorescence analysis demonstrated that implanted cells migrated from the lateral ventricle into the lesioned striatum and differentiated into striatal projection neurons. Array analysis showed a significant upregulation of GFR (Glial cell line-derived neurotrophic factor receptor) alpha-1, Adiponectin/Acrp30, basic-fibroblast growth factors, MIP-1 (Macrophage-inflammatory protein) alpha and leptin, as well as downregulation of cytokine-induced neutrophil chemoattractant-3 in striatum after transplantatation of induced pluripotent stem cells in comparison with the quinolinic acid -treated rats. The findings in this work indicate that transplantation of induced pluripotent stem cells is a promising therapeutic candidate for HD. © 2016 British Neuropathological Society.

Identification of a novel MIP frameshift mutation associated with congenital cataract in a Chinese family by whole-exome sequencing and functional analysis.

PubMed

Long, Xigui; Huang, Yanru; Tan, Hu; Li, Zhuo; Zhang, Rui; Linpeng, Siyuan; Lv, Weigang; Cao, Yingxi; Li, Haoxian; Liang, Desheng; Wu, Lingqian

2018-04-26

To detect the underlying pathogenesis of congenital cataract in a four-generation Chinese family. Whole-exome sequencing (WES) of family members (III:4, IV:4, and IV:6) was performed. Sanger sequencing and bioinformatics analysis were subsequently conducted. Full-length WT-MIP or K228fs-MIP fused to HA markers at the N-terminal was transfected into HeLa cells. Next, quantitative real-time PCR, western blotting and immunofluorescence confocal laser scanning were performed. The age of onset for nonsyndromic cataracts in male patients was by 1-year old, earlier than for female patients, who exhibited onset at adulthood. A novel c.682_683delAA (p.K228fs230X) mutation in main intrinsic protein (MIP) cosegregated with the cataract phenotype. The instability index and unfolded states for truncated MIP were predicted to increase by bioinformatics analysis. The mRNA transcription level of K228fs-MIP was reduced compared with that of WT-MIP, and K228fs-MIP protein expression was also lower than that of WT-MIP. Immunofluorescence images showed that WT-MIP principally localized to the plasma membrane, whereas the mutant protein was trapped in the cytoplasm. Our study generated genetic and primary functional evidence for a novel c.682_683delAA mutation in MIP that expands the variant spectrum of MIP and help us better understand the molecular basis of cataract.

Identification of markers for quiescent pancreatic stellate cells in the normal human pancreas.

PubMed

Nielsen, Michael Friberg Bruun; Mortensen, Michael Bau; Detlefsen, Sönke

2017-10-01

Pancreatic stellate cells (PSCs) play a central role as source of fibrogenic cells in pancreatic cancer and chronic pancreatitis. In contrast to quiescent hepatic stellate cells (qHSCs), a specific marker for quiescent PSCs (qPSCs) that can be used in formalin-fixed and paraffin embedded (FFPE) normal human pancreatic tissue has not been identified. The aim of this study was to identify a marker enabling the identification of qPSCs in normal human FFPE pancreatic tissue. Immunohistochemical (IHC), double-IHC, immunofluorescence (IF) and double-IF analyses were carried out using a tissue microarray consisting of cores with normal human pancreatic tissue. Cores with normal human liver served as control. Antibodies directed against adipophilin, α-SMA, CD146, CRBP-1, cytoglobin, desmin, GFAP, nestin, S100A4 and vinculin were examined, with special emphasis on their expression in periacinar cells in the normal human pancreas and perisinusoidal cells in the normal human liver. The immunolabelling capacity was evaluated according to a semiquantitative scoring system. Double-IF of the markers of interest together with markers for other periacinar cells was performed. Moreover, the utility of histochemical stains for the identification of human qPSCs was examined, and their ultrastructure was revisited by electron microscopy. Adipophilin, CRBP-1, cytoglobin and vinculin were expressed in qHSCs in the liver, whereas cytoglobin and adipophilin were expressed in qPSCs in the pancreas. Adipophilin immunohistochemistry was highly dependent on the preanalytical time interval (PATI) from removal of the tissue to formalin fixation. Cytoglobin, S100A4 and vinculin were expressed in periacinar fibroblasts (FBs). The other examined markers were negative in human qPSCs. Our data indicate that cytoglobin and adipophilin are markers of qPSCs in the normal human pancreas. However, the use of adipophilin as a qPSC marker may be limited due to its high dependence on optimal PATI. Cytoglobin, on the other hand, is a sensitive marker for qPSCs but is expressed in FBs as well.

In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses.

PubMed

Kohyama, Moeko; Yabuki, Akira; Ochiai, Kenji; Nakamoto, Yuya; Uchida, Kazuyuki; Hasegawa, Daisuke; Takahashi, Kimimasa; Kawaguchi, Hiroaki; Tsuboi, Masaya; Yamato, Osamu

2016-03-31

GM1 and GM2 gangliosidoses are progressive neurodegenerative lysosomal storage diseases resulting from the excessive accumulation of GM1 and GM2 gangliosides in the lysosomes, respectively. The diagnosis of gangliosidosis is carried out based on comprehensive findings using various types of specimens for histological, ultrastructural, biochemical and genetic analyses. Therefore, the partial absence or lack of specimens might have resulted in many undiagnosed cases. The aim of the present study was to establish immunohistochemical and immunofluorescent techniques for the auxiliary diagnosis of canine and feline gangliosidoses, using paraffin-embedded brain specimens stored for a long period. Using hematoxylin and eosin staining, cytoplasmic accumulation of pale to eosinophilic granular materials in swollen neurons was observed in animals previously diagnosed with GM1 or GM2 gangliosidosis. The immunohistochemical and immunofluorescent techniques developed in this study clearly demonstrated the accumulated material to be either GM1 or GM2 ganglioside. Immunohistochemical and immunofluorescent techniques using stored paraffin-embedded brain specimens are useful for the retrospective diagnosis of GM1 and GM2 gangliosidoses in dogs and cats.

Increased serum anti-N-methyl-D-aspartate receptor antibody immunofluorescence in psychiatric patients with past catatonia

PubMed Central

Lin, Chin-Chuen; Hung, Yi-Yung; Tsai, Meng-Chang

2017-01-01

Objective Anti-N-methyl-D-aspartate receptor (NMDAR) antibody was thought to be the cause of anti-NMDAR encephalitis, with manifestations similar to catatonia and schizophrenia. Anti-NMDAR antibody in neuropsychiatric patients who had catatonia before were investigated in a follow-up evaluation. The intensity of antibody immunofluorescence was quantified and compared with healthy controls. Method Nineteen patients (eight males and eleven females) agreed to be followed-up. Thirteen had the diagnosis of schizophrenia, two had the diagnosis of major depressive disorder, two had bipolar disorder, one had postpartum depression, and one had herpes simplex encephalitis. No patient had catatonia during the follow-up. Nineteen sex-matched healthy controls were recruited. Results Using Mann-Whitney U test, patients had greater intensity of anti-NMDAR antibody immunofluorescence than the healthy controls (121,979 ± 86,526 vs. 47,692 ± 26,102, p = 0.003). No correlation was found between immunofluorescence intensity and catatonia scales or symptom severity scores. Neuropsychiatric patients with past catatonia showed greater anti-NMDAR antibody response than the healthy controls. Conclusion NMDAR dysfunction might play a role in the mechanism underlying catatonia. Further studies are needed to confirm this finding. PMID:29073246

Increased serum anti-N-methyl-D-aspartate receptor antibody immunofluorescence in psychiatric patients with past catatonia.

PubMed

Lin, Chin-Chuen; Hung, Yi-Yung; Tsai, Meng-Chang; Huang, Tiao-Lai

2017-01-01

Anti-N-methyl-D-aspartate receptor (NMDAR) antibody was thought to be the cause of anti-NMDAR encephalitis, with manifestations similar to catatonia and schizophrenia. Anti-NMDAR antibody in neuropsychiatric patients who had catatonia before were investigated in a follow-up evaluation. The intensity of antibody immunofluorescence was quantified and compared with healthy controls. Nineteen patients (eight males and eleven females) agreed to be followed-up. Thirteen had the diagnosis of schizophrenia, two had the diagnosis of major depressive disorder, two had bipolar disorder, one had postpartum depression, and one had herpes simplex encephalitis. No patient had catatonia during the follow-up. Nineteen sex-matched healthy controls were recruited. Using Mann-Whitney U test, patients had greater intensity of anti-NMDAR antibody immunofluorescence than the healthy controls (121,979 ± 86,526 vs. 47,692 ± 26,102, p = 0.003). No correlation was found between immunofluorescence intensity and catatonia scales or symptom severity scores. Neuropsychiatric patients with past catatonia showed greater anti-NMDAR antibody response than the healthy controls. NMDAR dysfunction might play a role in the mechanism underlying catatonia. Further studies are needed to confirm this finding.

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

PubMed

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Increased expression of T cell immunoglobulin and mucin domain 3 aggravates brain inflammation via regulation of the function of microglia/macrophages after intracerebral hemorrhage in mice.

PubMed

Xu, ChangJun; Wang, Tao; Cheng, Si; Liu, YuGuang

2013-12-01

Microglia/macrophages are known to play important roles in initiating brain inflammation after spontaneous intracerebral hemorrhage (ICH). T cell immunoglobulin and mucin domain-3 (Tim-3) have been proven to play a critical part in several inflammatory diseases through regulation of both adaptive and innate immune responses. Tim-3 can be expressed by microglia/macrophages and regulates their function in the innate immune response. However, the effect of Tim-3 on inflammatory responses following ICH is unclear. In this study, we investigated Tim-3 expression, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and brain water content in peri-hematomal brain tissue at 12 hours and at 1, 3, 5, and 7 days post-ICH in wild type (WT) ICH and Tim-3-/- ICH mice. The numbers of Tim-3 positive cells,astrocytes, neutrophils and microglia/macrophages were detected using immunofluorescence staining. Cytokines were measured by ELISA. Double immunofluorescence labeling was performed to identify the cellular source of Tim-3 expression. Mouse neurological deficit scores were assessed through animal behavior. Expression of Tim-3 increased early in mouse peri-hematomal brain tissue after autologous blood injection, peaked at day 1, and was positively correlated with the concentrations of TNF-α, IL-1β, and brain water content. Tim-3 was predominantly expressed in microglia/macrophages. Compared with WT mice, Tim-3-/- mice had reduced ICH-induced brain inflammation with decreased TNF-α and IL-1β, cerebral edema and neurological deficit scores. Moreover, Tim-/- inhibited activation of microglia/macrophages. The number of activated microglia/macrophages in Tim-3-/- ICH mice was much lower than that in WT ICH mice. Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH, and may be a potential treatment target.

Expression of T-box transcription factors 2, 4 and 5 is decreased in the branching airway mesenchyme of nitrofen-induced hypoplastic lungs.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2017-02-01

Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major therapeutic challenge in newborns with congenital diaphragmatic hernia (CDH). T-box transcription factors (Tbx) have been identified as key components of the gene network that regulates fetal lung development. Tbx2, Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung, regulating the process of lung branching morphogenesis. Furthermore, lungs of Tbx2-, Tbx4- and Tbx5-deficient mice are hypoplastic and exhibit decreased lung branching, similar to PH in human CDH. We hypothesized that the expression of Tbx2, Tbx4 and Tbx5 is decreased in the branching airway mesenchyme of hypoplastic rat lungs with nitrofen-induced CDH. Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetuses were killed on D15, D18 and D21, and dissected lungs were divided into control and nitrofen-exposed specimens. Pulmonary gene expression of Tbx2, Tbx4 and Tbx5 was investigated by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for Tbx2, Tbx4 and Tbx5 was combined with the mesenchymal marker Fgf10 to assess protein expression and localization in branching airway tissue. Relative mRNA levels of Tbx2, Tbx4 and Tbx5 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Tbx2, Tbx4 and Tbx5 in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Decreased expression of Tbx2, Tbx4 and Tbx5 in the pulmonary mesenchyme during fetal lung development may lead to a decrease or arrest of airway branching, thus contributing to PH in the nitrofen-induced CDH model.

Decreased Expression of Integrin Subunits α3, α6, and α8 in the Branching Airway Mesenchyme of Nitrofen-Induced Hypoplastic Lungs.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2018-02-01

 Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major cause of neonatal mortality in newborns with congenital diaphragmatic hernia (CDH). Integrin-mediated cell-matrix interactions play an essential role in the fetal lung mesenchyme by stimulating branching morphogenesis. Mice lacking integrin subunits α3 (Itga3) and α6 (Itga6) exhibit severe PH. Furthermore, Itga8-knockout mice show defective airway branching, suggesting that Itga3, Itga6, and Itga8 are crucial for fetal lung development. We hypothesized that expression of Itga3, Itga6, and Itga8 is decreased in the branching airway mesenchyme of hypoplastic rat lungs in the nitrofen-induced CDH model.  Time-mated rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D15, D18, and D21, and dissected lungs were divided into control and nitrofen-exposed specimens ( n  = 12 per time-point and group, respectively). Pulmonary gene expression of Itga3, Itga6, and Itga8 was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double-staining for Itga3, Itga6, and Itga8 was combined with the mesenchymal marker Fgf10 to evaluate protein expression and localization in branching airway tissue.  Relative mRNA expression of Itga3, Itga6, and Itga8 was significantly decreased in lungs of nitrofen-exposed fetuses on D15, D18, and D21 compared with controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Itga3, Itga6, and Itga8 mainly in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18, and D21 compared with controls.  Decreased expression of Itga3, Itga6, and Itga8 in the pulmonary mesenchyme may lead to disruptions in airway branching morphogenesis, thus contributing to PH in the nitrofen-induced CDH model. Georg Thieme Verlag KG Stuttgart · New York.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

MLN8054, A Small Molecule Inhibitor of Aurora Kinase A, Sensitizes Androgen-Resistant Prostate Cancer to Radiation;Aurora kinase A; MLN8054; Prostate cancer; Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moretti, Luigi; Department of Radiation Oncology, Institut Jules Bordet, Universite Libre de Bruxelles, Brussels; Niermann, Kenneth

2011-07-15

Purpose: To determine whether MLN8054, an Aurora kinase A (Aurora-A) inhibitor causes radiosensitization in androgen-insensitive prostate cancer cells in vitro and in vivo. Methods and Materials: In vitro studies consisted of culturing PC3 and DU145 prostate cancer cells and then immunoblotting Aurora A and phospho-Aurora A after radiation and/or nocodazole with MLN8054. Phases of the cell cycle were measured with flow cytometry. PC3 and DU145 cell lines were measured for survival after treatment with MLN8054 and radiation. Immunofluorescence measured {gamma}-H2AX in the PC3 and DU145 cells after treatment. In vivo studies looked at growth delay of PC3 tumor cells inmore » athymic nude mice. PC3 cells grew for 6 to 8 days in mice treated with radiation, MLN8054, or combined for 7 more days. Tumors were resected and fixed on paraffin and stained for von Willebrand factor, Ki67, and caspase-3. Results: In vitro inhibition of Aurora-A by MLN8054 sensitized prostate cancer cells, as determined by dose enhancement ratios in clonogenic assays. These effects were associated with sustained DNA double-strand breaks, as evidenced by increased immunofluorescence for {gamma}-H2AX and significant G2/M accumulation and polyploidy. In vivo, the addition of MLN8054 (30 mg/kg/day) to radiation in mouse prostate cancer xenografts (PC3 cells) significantly increased tumor growth delay and apoptosis (caspase-3 staining), with reduction in cell proliferation (Ki67 staining) and vascular density (von Willebrand factor staining). Conclusion: MLN8054, a novel small molecule Aurora-A inhibitor showed radiation sensitization in androgen-insensitive prostate cancer in vitro and in vivo. This warrants the clinical development of MLN8054 with radiation for prostate cancer patients.« less

Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

PubMed

Forsey, T; Darougar, S

1980-02-01

A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital herpes infections showed that the test can differentiate antibodies to the infecting serotypes. Specimens of whole blood, taken by fingerprick, and eye secretions, both collected on cellulose sponges, could be tested by indirect micro-immunofluorescence. Anti-herpes IgG, IgM, and IgA can also be detected.

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

PubMed

Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of sertraline on proliferation and neurogenic differentiation of human adipose-derived stem cells

PubMed Central

Razavi, Shahnaz; Jahromi, Maliheh; Amirpour, Nushin; Khosravizadeh, Zahra

2014-01-01

Background: Antidepressant drugs are commonly employed for anxiety and mood disorders. Sertraline is extensively used as antidepressant in clinic. In addition, adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human adipose-derived stem cells (hADSCs) may be useful for autologous transplantation. Materials and Methods: In the present study, we assessed the effect of antidepressant drug Sertraline on the proliferation and neurogenic differentiation of hADSCs using MTT assay and immunofluorescence technique respectively. Results: MTT assay analysis showed that 0.5 μM Sertraline significantly increased the proliferation rate of hADSCs induced cells (P < 0.05), while immunofluorescent staining indicated that Sertraline treatment during neurogenic differentiation could be decreased the percentage of glial fibrillary acidic protein and Nestin-positive cells, but did not significantly effect on the percentage of MAP2 positive cells. Conclusion: Overall, our data show that Sertraline can be promoting proliferation rate during neurogenic differentiation of hADSCs after 6 days post-induction, while Sertraline inhibits gliogenesis of induced hADSCs. PMID:24800186

Integration of co-localized glandular morphometry and protein biomarker expression in immunofluorescent images for prostate cancer prognosis

NASA Astrophysics Data System (ADS)

Scott, Richard; Khan, Faisal M.; Zeineh, Jack; Donovan, Michael; Fernandez, Gerardo

2015-03-01

Immunofluorescent (IF) image analysis of tissue pathology has proven to be extremely valuable and robust in developing prognostic assessments of disease, particularly in prostate cancer. There have been significant advances in the literature in quantitative biomarker expression as well as characterization of glandular architectures in discrete gland rings. However, while biomarker and glandular morphometric features have been combined as separate predictors in multivariate models, there is a lack of integrative features for biomarkers co-localized within specific morphological sub-types; for example the evaluation of androgen receptor (AR) expression within Gleason 3 glands only. In this work we propose a novel framework employing multiple techniques to generate integrated metrics of morphology and biomarker expression. We demonstrate the utility of the approaches in predicting clinical disease progression in images from 326 prostate biopsies and 373 prostatectomies. Our proposed integrative approaches yield significant improvements over existing IF image feature metrics. This work presents some of the first algorithms for generating innovative characteristics in tissue diagnostics that integrate co-localized morphometry and protein biomarker expression.

Direct immunofluorescence of normal skin in rheumatoid arthritis.

PubMed

Fitzgerald, O M; Barnes, L; Woods, R; McHugh, L; Barry, C; O'Loughlin, S

1985-11-01

The clinical significance of previously described immunoglobulin and complement deposition in the superficial dermal vessel walls of patients with rheumatoid arthritis is unknown. In the present study, skin biopsies were obtained from the normal forearm and buttock of 48 unselected patients with rheumatoid arthritis and were examined by direct immunofluorescence (IF) for the presence of immunoglobulin (IgG,A,M) and complement (C3) in the vessel walls. Deposits of C3, IgM or IgG were detected in 10 patients. Five patients had deposits at the forearm sample alone, four patients had deposits at both biopsy sites, while one patient was positive at the buttock alone. Clinical features were similar in patients with and without vessel IF. However, patients with IF were significantly more seropositive with lower levels of complement and raised levels of serum IgA and IgM. There was also an increased level of circulating IgG immune complexes in these patients. Further analysis following exclusion of seronegative patients revealed similar results. This study suggests that the presence of vessel IF identifies a subgroup of patients who have evidence of more severe immunological disturbance.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

PubMed Central

Zimmermann, K; Herget, T; Salbaum, J M; Schubert, W; Hilbich, C; Cramer, M; Masters, C L; Multhaup, G; Kang, J; Lemaire, H G

1988-01-01

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates. Images PMID:2896589

Detection of Osteopontin in the pericyst of human hepatic Echinococcus granulosus.

PubMed

Peng, Xinyu; Li, Jianhui; Wu, Xiangwei; Zhang, Shijie; Niu, Jianhua; Chen, Xiaoping; Yao, Jin; Sun, Hong

2006-12-01

It aims at investigating the expression and distribution of the Osteopontin (OPN) in the pericyst of human hepatic Echinococcus granulosus and their related significances. Sixty pericysts excised by "sub-adventitial cystectomy" were studied. OPN was detected in 80% (48/60) of cysts by Western blotting and distributed in the side of "exocyst" layer directing to the parasite, also macrophages were identified in the vicinity of OPN by immunohistochemistry staining. The coexpression of OPN and CD68 was observed by immunofluorescence double labeling and analyzed by Image-Pro Plus 5.1; with special stain techniques, variable degrees of calcium deposits were observed in 80% (48/60) cysts, and the calcium deposits concurrencely found with the OPN expression. The selective distribution of OPN, calcium in the "exocyst" provides a new pathological evidence for the "sub-adventitial cystectomy" we developed. The pericyst of hepatic E. granulosus consists of two detachable layers with different formative mechanisms: the "exocyst" layer directing towards the cyst of parasite was the result of granulomatous reaction; also the results suggest OPN is one regulator in the granulomatous reaction and calcification of "exocyst".

The G-protein-coupled estrogen receptor (GPER) is expressed in normal human ovaries and is upregulated in ovarian endometriosis and pelvic inflammatory disease involving the ovary.

PubMed

Heublein, Sabine; Lenhard, Miriam; Vrekoussis, Thomas; Schoepfer, Jutta; Kuhn, Christina; Friese, Klaus; Makrigiannakis, Antonis; Mayr, Doris; Jeschke, Udo

2012-11-01

Estrogens play a crucial role in maintaining ovarian function. Deregulation of estrogen signals is associated with fertility-impairing disorders. The aim of this study was to investigate whether the G-protein-coupled estrogen receptor (GPER) is present in the human ovary. Additionally, we  analyzed the folliculogenesis and ovarian endometriosis in GPER expression. Seventy-nine patients (ovarian endometriosis, n = 26; ovarian pelvic inflammatory disease [PID], n = 10; normal ovaries/endometrium, n = 30/13) were analyzed by immunohistochemistry. Normal ovaries were also assessed by real-time polymerase chain reaction and double immunofluorescence. The most intense expression of GPER was noted in the ovarian surface epithelium. Theca cells and oocytes were also significantly positive. Expression of GPER was more frequent in mature follicles/oocytes than in primordial ones, implying that GPER could be a selector during folliculogenesis. Moreover, GPER was upregulated in ovarian endometriosis and PID. Overexpression of GPER in both inflammation and endometriosis affecting the ovary may prove useful in explaining/predicting the main endometriosis-related symptoms.

[Epithelial intestine cells transdifferentiate into bladder urothelium in experiments in vivo].

PubMed

Popov, B K; Zaĭchik, A M; Bud'ko, M B; Zlobina, O V; Tolkunova, E N; Zhidkova, O V; Petrov, N S

2011-01-01

The autoplastic surgery by intestine tissue has been used for reconstructive therapy of the urinary tract since the middle of the last century; however, cell mechanisms of the urothelium engraftment are still obscure. Intestine stem cells possess plasticity and presumably enable after the autoplastic surgery to transdifferentiate into mature cells of urinary tract. Using the preliminary developed in vivo model for evaluation of somatic cells transdifferentiation into urothelium, we have found that the epithelial intestine cells producing Gfp transdifferentiate into the cryoinjured bladder urothelium of the syngenetic C57BL mice. Gfp was detected in the bladder tissue of mice-recipients using reverted polymerase chain reaction, primary fluorescence and immunofluorescence, while colocalization of the Gfp and Her-4 revealing similar to urothelium staining pattern was demonstrated in a few urothelium cells by double immunohistochemical staining of the bladder tissue with specific antibodies. The results obtained suggest that epithelial intestine cells enable to transdifferentiate into bladder urothelium, however the transdifferentiation level is low and presumably can not provide full functional urothelium engraftment in the case of autoplastic bladder surgery by intestine tissue.

Lymphatic vessels correlate closely with inflammation index in alkali burned cornea.

PubMed

Yan, Hao; Qi, Chaoxiu; Ling, Shiqi; Li, Weihua; Liang, Linyi

2010-08-01

To study the relationship between corneal lymphangiogenesis and inflammation in alkali burned corneas. Rat corneal lymphatic and blood vessels were labeled and distinguished by whole mount immunofluorescence and 5'-nase-alkaline phosphatase (5'-NA-ALP) double enzyme-histochemistry. Then, lymphatic vessel areas (LVA) and lymphatic vessel counting (LVC) were examined. Corneal inflammation was evaluated by inflammation index (IF) grading, histopathology, electron microscope, and polymorphonuclear leukocyte (PMN) infiltration. The relationship between LVC, LVA, IF, and PMN was examined, respectively. In addition, corneal lymphatic vessels of eleven human alkali burned corneas were examined by lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry. Corneal lymphangiogenesis occurred on Day 3, reached the peak at the end of two weeks, and disappeared five weeks after alkaline burns. Both LVA and LVC were strongly and positively correlated with IF after corneal alkaline burns. However, the relationship between LVC and PMN, between LVA and PMN were significant but converse. Among eleven human alkali burned corneas, corneal lymphangiogenesis was present in three corneas. Corneal lymphagiogenesis develops after alkaline burns and correlates closely with corneal inflammation.

Effect of cortisol on gonadotropin inhibitory hormone (GnIH) in the cinnamon clownfish, Amphiprion melanopus.

PubMed

Choi, Young Jae; Habibi, Hamid R; Kil, Gyung-Suk; Jung, Min-Min; Choi, Cheol Young

2017-04-01

Hypothalamic peptides, gonadotropin-releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH), play pivotal roles in the control of reproduction and gonadal maturation in fish. In the present study we tested the possibility that stress-mediated reproductive dysfunction in teleost may involve changes in GnRH and GnIH activity. We studied expression of brain GnIH, GnIH-R, seabream GnRH (sbGnRH), as well as circulating levels of follicle stimulating hormone (FSH), and luteinizing hormone (LH) in the cinnamon clownfish, Amphiprion melanopus. Treatment with cortisol increased GnIH mRNA level, but reduced sbGnRH mRNA and circulating levels of LH and FSH in cinnamon clownfish. Using double immunofluorescence staining, we found expression of both GnIH and GnRH in the diencephalon region of cinnamon clownfish brain. These findings support the hypothesis that cortisol, an indicator of stress, affects reproduction, in part, by increasing GnIH in cinnamon clownfish which contributes to hypothalamic suppression of reproductive function in A. melanopus, a protandrous hermaphroditic fish. Copyright © 2017 Elsevier Inc. All rights reserved.

The Inhibitory Effects of Npas4 on Seizures in Pilocarpine-Induced Epileptic Rats

PubMed Central

Guo, Jiamei; Yang, Guang; Long, Xianghua; Hu, Rong; Shen, Wenjing; Wang, Xuefeng; Zeng, Kebin

2014-01-01

To explore the effects of neuronal Per-Arnt-Sim domain protein 4 (Npas4) on seizures in pilocarpine-induced epileptic rats, Npas4 expression was detected by double-label immunofluorescence, immunohistochemistry, and Western blotting in the brains of pilocarpine-induced epileptic model rats at 6 h, 24 h, 72 h, 7 d, 14 d, 30 d, and 60 d after status epilepticus. Npas4 was localized primarily in the nucleus and in the cytoplasm of neurons. The Npas4 protein levels increased in the acute phase of seizures (between 6 h and 72 h) and decreased in the chronic phases (between 7 d and 60 d) in the rat model. Npas4 expression was knocked down by specific siRNA interference. Then, the animals were treated with pilocarpine, and the effects on seizures were evaluated on the 7th day. The onset latencies of pilocarpine-induced seizures were decreased, while the seizure frequency, duration and attack rate increased in these rats. Our study indicates that Npas4 inhibits seizure attacks in pilocarpine-induced epileptic rats. PMID:25536221

The inhibitory effects of Npas4 on seizures in pilocarpine-induced epileptic rats.

PubMed

Wang, Dan; Ren, Min; Guo, Jiamei; Yang, Guang; Long, Xianghua; Hu, Rong; Shen, Wenjing; Wang, Xuefeng; Zeng, Kebin

2014-01-01

To explore the effects of neuronal Per-Arnt-Sim domain protein 4 (Npas4) on seizures in pilocarpine-induced epileptic rats, Npas4 expression was detected by double-label immunofluorescence, immunohistochemistry, and Western blotting in the brains of pilocarpine-induced epileptic model rats at 6 h, 24 h, 72 h, 7 d, 14 d, 30 d, and 60 d after status epilepticus. Npas4 was localized primarily in the nucleus and in the cytoplasm of neurons. The Npas4 protein levels increased in the acute phase of seizures (between 6 h and 72 h) and decreased in the chronic phases (between 7 d and 60 d) in the rat model. Npas4 expression was knocked down by specific siRNA interference. Then, the animals were treated with pilocarpine, and the effects on seizures were evaluated on the 7th day. The onset latencies of pilocarpine-induced seizures were decreased, while the seizure frequency, duration and attack rate increased in these rats. Our study indicates that Npas4 inhibits seizure attacks in pilocarpine-induced epileptic rats.

Bortezomib sensitizes esophageal squamous cancer cells to radiotherapy by suppressing the expression of HIF-1α and apoptosis proteins.

PubMed

Wang, Di; Qin, Qin; Jiang, Qin-Juan; Wang, Da-Fei

2016-04-13

Radiation therapy is a typical treatment for esophageal squamous cell carcinoma (ESCC), especially middle and upper segment esophagus, and inoperable patients. However, how to promote radiation sensitivity in radio-resistant cancer cells is a conundrum. Here, our study investigated the radiosensitizing effect of bortezomib, a specific and reversible dipeptide boronic acid analog, in ESCC cells. Human esophageal squamous carcinoma cell lines Eca109 and TE-13 were exposed to hypoxia and/or ionizing radiation (IR) with or without treatment of bortezomib. Cell proliferation assay was performed with CCK8. Cell apoptosis and cell cycle assay were performed with flow cytometry. The radiosensitization effect of was assessed by clonogenic survival and progression of tumor xenograft. The expression of HIF-1α, VEGF, and apoptosis proteins was evaluated by Western blot. Radiation-induced DNA double strand break and homologous recombination repair were assessed by immunofluorescence. Our results show that bortezomib efficiently radiosensitizes ESCC cells by decreasing the expression of HIF- 1α and VEGF, inducing apoptosis by activating caspase, and delaying DNA damage repair after radiation.

Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury.

PubMed

Eto, Hideyuki; Miyata, Masaaki; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

2006-03-10

Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 microg/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescence staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

PubMed

Ochoa, Gina H; Clark, Ying Mei; Matsumoto, Brian; Torres-Ruiz, Jose A; Robles, Laura J

2002-02-01

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.

A study of the aetiology of gastritis following gastric surgery. I. Immunofluorescent studies of the gastric mucosa.

PubMed Central

Chapel, H M; Hoare, A M

1979-01-01

The gastritis which follows surgical trauma is probably not of autoimmune origin. Although identical with the gastritis of pernicious anaemia (PA) upon routine histological examination, immunofluorescent examination of post-operative gastritis differs in that IgA-containing plasma cells alone are found, in contrast to the predominantly IgG-containing plasma cells in PA. PMID:389496

Mesenchymal stem cells reside in a vascular niche in the decidua basalis and are absent in remodelled spiral arterioles.

PubMed

Kusuma, G D; Manuelpillai, U; Abumaree, M H; Pertile, M D; Brennecke, S P; Kalionis, B

2015-03-01

Maternal decidua basalis tissue attached to the placenta following delivery is a source of decidual mesenchymal stem cells (DMSCs). The in vitro characteristics of DMSCs have been partly defined but their in vivo function(s) are poorly understood. The anatomic location, or niche, provides clues regarding potential in vivo function(s) of DMSCs, but the niche has not been described. Cells were isolated from the decidua basalis and flow cytometric analyses showed the expected phenotypic profile for MSC cell surface markers. In vitro, the cells differentiated into adipocytes, osteocytes, and chondrocytes. DMSCs were then stained with antibodies by immunofluorescence detection. Immunocytochemistry revealed that DMSCs were positive for FZD-9, STRO-1, 3G5, and α-SMA as expected and lacked expression of vWF and Ck7. Fluorescence in situ hybridization analysis showed the cultured cells were of maternal origin. Immunofluorescence was carried out on placental bed biopsies using the FZD-9, STRO-1, 3G5, and α-SMA antibodies. DMSCs were located in the vascular niche in decidua basalis. Immunofluorescence with antibodies to FZD-9, Ck7 and vWF revealed DMSCs in the vascular niche surrounding intact non-transformed spiral arterioles but DMSCs were absent in fully transformed spiral arterioles. Spiral arteriole remodelling is a critical feature of human pregnancy. The DMSC niche was investigated in fully transformed and non-transformed spiral arterioles. DMSCs have not been previously implicated in spiral arteriole remodelling. The absence of DMSCs around fully transformed spiral arterioles suggests they are a target for replacement or destruction by invading placental extravillous trophoblast cells, which carry out spiral arteriole remodelling. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel serine protease predominately expressed in macrophages.

PubMed Central

Chen, Cailin; Darrow, Andrew L; Qi, Jian-Shen; D'Andrea, Michael R; Andrade-Gordon, Patricia

2003-01-01

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders. PMID:12795636

Lysyl oxidase‑like 2 is expressed in kidney tissue and is associated with the progression of tubulointerstitial fibrosis.

PubMed

Choi, Sung-Eun; Jeon, Nara; Choi, Hoon Young; Shin, Jae Il; Jeong, Hyeon Joo; Lim, Beom Jin

2017-09-01

Tubulointerstitial fibrosis is a common end point of chronic kidney diseases, and preventing its progression is key to avoiding renal failure. Transforming growth factor‑β (TGF‑β) and associated molecules promote tubulointerstitial fibrosis; however, effective therapies targeting these molecules have yet to be developed. Lysyl oxidase‑like 2 (LOXL2), which is involved in invasive growth and metastasis of malignant neoplasms, has recently been reported to serve a key role in hepatic and pulmonary fibrosis. However, little is currently known regarding LOXL2 expression in the kidney and its involvement in tubulointerstitial fibrosis. The present study evaluated LOXL2 expression in human and mouse kidney tissues, as well as in cultured renal cells. LOXL2 protein expression was detected in glomerular capillary loops and tubular epithelial cells in human and mouse kidneys. Glomerular LOXL2 was localized to the cytoplasm of podocytes, as determined by double immunofluorescence microscopy using a podocyte marker (synaptopodin). This result was supported by western blot analysis, which demonstrated that LOXL2 protein expression is present in cultured human podocytes and HK‑2 human proximal tubular cells. In addition, the mRNA and protein expression levels of LOXL2 were higher in a mouse model of tubulointerstitial fibrosis compared with in control mice. In addition, immunohistochemistry results demonstrated that LOXL2 is present in the fibrous interstitium and infiltrating mononuclear cells in a mouse model of tubulointerstitial fibrosis. The present study demonstrated that LOXL2 is expressed in compartments of renal tissue, where it appears to contribute to the progression of tubulointerstitial fibrosis.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

PubMed

Nasu, Masanori; Nakahara, Taka; Tominaga, Noriko; Tamaki, Yuichi; Ide, Yoshiaki; Tachibana, Toshiaki; Ishikawa, Hiroshi

2013-03-01

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/Akt/mTOR signaling pathway.

PubMed

Wang, Shanshan S; Chen, Yuhan H; Chen, Ning; Wang, Lijun J; Chen, Dexi X; Weng, Honglei L; Dooley, Steven; Ding, Huiguo G

2017-03-23

Hydrogen sulfide (H 2 S), in its gaseous form, plays an important role in tumor carcinogenesis. This study investigated the effects of H 2 S on the cell biological functions of hepatocellular carcinoma (HCC). HCC cell lines, HepG2 and HLE, were treated with NaHS, a donor of H 2 S, and rapamycin, a classic autophagy inducer, for different lengths of time. Western blotting, immunofluorescence, transmission electron microscopy (TEM), scratch assay, CCK-8 and flow cytometric analysis were carried out to examine the effects of H 2 S on HCC autophagy, cell behavior and PI3K/Akt/mTOR signaling. Treatment with NaHS upregulated expression of LC3-II and Atg5, two autophagy-related proteins, in HepG2 and HLE cells. TEM revealed increased numbers of intracellular double-membrane vesicles in those cells treated with NaHS. Like rapamycin, NaHS also significantly inhibited expression of p-PI3K, p-Akt and mTOR proteins in HCC cells. Interestingly, the expression of LC3-II was further increased when the cells were treated with NaHS together with rapamycin. In addition, NaHS inhibited HCC cell migration, proliferation and cell division. These findings show that H 2 S can induce HCC cell apoptosis. The biological function of the gasotransmitter H 2 S in HCC cells was enhanced by the addition of rapamycin. Hydrogen sulfide influences multiple biological functions of HCC cells through inhibiting the PI3K/Akt/mTOR signaling pathway.

Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

PubMed

Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-Lin; Liu, Ke; Shang, Zheng-Jun

2014-10-15

Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Methamphetamine induces heme oxygenase-1 expression in cortical neurons and glia to prevent its toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Y.-N.; Wu, C.-H.; Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan 114

2009-11-01

The impairment of cognitive and motor functions in humans and animals caused by methamphetamine (METH) administration underscores the importance of METH toxicity in cortical neurons. The heme oxygenase-1 (HO-1) exerts a cytoprotective effect against various neuronal injures; however, it remains unclear whether HO-1 is involved in METH-induced toxicity. We used primary cortical neuron/glia cocultures to explore the role of HO-1 in METH-induced toxicity. Exposure of cultured cells to various concentrations of METH (0.1, 0.5, 1, 3, 5, and 10 mM) led to cytotoxicity in a concentration-dependent manner. A METH concentration of 5 mM, which caused 50% of neuronal death andmore » glial activation, was chosen for subsequent experiments. RT-PCR and Western blot analysis revealed that METH significantly induced HO-1 mRNA and protein expression, both preceded cell death. Double and triple immunofluorescence staining further identified HO-1-positive cells as activated astrocytes, microglia, and viable neurons, but not dying neurons. Inhibition of the p38 mitogen-activated protein kinase pathway significantly blocked HO-1 induction by METH and aggravated METH neurotoxicity. Inhibition of HO activity using tin protoporphyrine IX significantly reduced HO activity and exacerbated METH neurotoxicity. However, prior induction of HO-1 using cobalt protoporphyrine IX partially protected neurons from METH toxicity. Taken together, our results suggest that induction of HO-1 by METH via the p38 signaling pathway may be protective, albeit insufficient to completely protect cortical neurons from METH toxicity.« less

Morphometric alterations of Golgi apparatus in Alzheimer's disease are related to tau hyperphosphorylation.

PubMed

Antón-Fernández, Alejandro; Aparicio-Torres, Guillermo; Tapia, Silvia; DeFelipe, Javier; Muñoz, Alberto

2017-01-01

The Golgi apparatus (GA) is a highly dynamic organelle, which is mainly involved in the post-translational processing and targeting of cellular proteins and which undergoes significant morphological changes in response to different physiological and pathological conditions. In the present study, we have analyzed the possible alterations of GA in neurons from the temporal neocortex and hippocampus of Alzheimer's disease (AD) patients, using double immunofluorescence techniques, confocal microscopy and 3D quantification techniques. We found that in AD patients, the percentage of temporal neocortical and CA1 hippocampal pyramidal neurons with a highly altered GA is much higher (approximately 65%) in neurons with neurofibrillary tangles (NFT) than in NFT-free neurons (approximately 6%). Quantitative analysis of the surface area and volume of GA elements in neurons revealed that, compared with NFT-free neurons, NFT-bearing neurons had a reduction of approximately one half in neocortical neurons and one third in CA1 neurons. In both regions, neurons with a pre-tangle stage of phospho-tau accumulation had surface area and GA volume values that were intermediate, that is, between those of NFT-free and NFT-bearing neurons. These findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA including fragmentation and a decrease in the surface area and volume of GA elements. These alterations likely impact the processing and trafficking of proteins, which might contribute to neuronal dysfunction in AD. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Detection of DNA Damage by Space Radiation in Human Fibroblast Cells Flown on the International Space Station

NASA Technical Reports Server (NTRS)

Wu, Honglu; Lu, Tao; Wong, Michael; Beno, Jonathan; Countryman, Stefanie; Stodieck, Louis; Karouia, Fathi; Zhang, Ye

2015-01-01

Although charged particles in space have been detected with radiation detectors on board spacecraft since the early discovery of the Van Allen Belt, reports on effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a small number of changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells fixed on Days 3 and 14 after reaching orbit were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used marker for DNA double strand breaks. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground control. Human fibroblast cells were also exposed to low dose rate gamma rays, as well as to protons and Fe ions. Comparison of the pattern and distribution of the foci after gamma ray and charged particle exposure to our flight results confirmed that the foci found in the flown cells were indeed induced by space radiation.

Nonessential role of beta3 and beta5 integrin subunits for efficient clearance of cellular debris after light-induced photoreceptor degeneration.

PubMed

Joly, Sandrine; Samardzija, Marijana; Wenzel, Andreas; Thiersch, Markus; Grimm, Christian

2009-03-01

During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light. Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins. Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1. Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

PubMed

Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

2016-01-01

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

Intracellular pathways following uptake of bevacizumab in RPE cells.

PubMed

Aboul Naga, Shereen Hassan; Dithmer, Michaela; Chitadze, Guranda; Kabelitz, Dieter; Lucius, Ralph; Roider, Johann; Klettner, Alexa

2015-02-01

The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative study of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus.

PubMed

Zhou, Tingting; Fang, Siyue; Li, Chunlei; Hua, Hong

2016-11-01

Pemphigus is one of the potentially fatal autoimmune blistering diseases. An early and accurate diagnosis is important for prognosis and therapy. It may be difficult to diagnosis based on clinical grounds alone. Direct and indirect immunofluorescence, enzyme-linked immunosorbent assay, the Tzanck smear test, or histopathology are all available for the diagnosis of pemphigus. However, there are no generally accepted diagnostic criteria for the diagnosis of this condition at present. To evaluate the diagnostic value of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test for the diagnosis of pemphigus in dental clinics. A single-center retrospective study was conducted, and the clinical data of 33 patients with pemphigus and 61 controls were collected and analyzed from the Department of Oral Medicine, Peking University School of Stomatology, during 2010-2014. The sensitivities and specificities of indirect immunofluorescence, enzyme-linked immunosorbent assay, and the Tzanck smear test were calculated and compared in two groups. Sensitivities for the Tzanck smear test, indirect immunofluorescence, and enzyme-linked immunosorbent assay were 96.7%, 84.8%, and 84.8%, respectively, whereas the specificities of these tests were 60%, 91.8%, and 96.7%, respectively. The serial tests for the Tzanck smear test and enzyme-linked immunosorbent assay showed 82% sensitivity and 98.7% specificity. The serial test for the Tzanck smear test and enzyme-linked immunosorbent assay may represent a simple, rapid, and reliable way to definitive diagnosis of pemphigus. It is recommended as a common test for the diagnosis of pemphigus in dental clinics. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs) Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study

PubMed Central

Brand, Michael; Sommer, Matthias; Ellmann, Stephan; Wuest, Wolfgang; May, Matthias S.; Eller, Achim; Vogt, Sabine; Lell, Michael M.; Kuefner, Michael A.; Uder, Michael

2015-01-01

Background Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT) and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs) as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes. Materials and Methods Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC) and Q 10). Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX. Results For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%), selenium (14%), vitamin E (12%), vitamin C (25%), NAC (43%) and Q 10 (18%) led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect. Conclusion Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes. PMID:25996998

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

PubMed

Wagner, J M; Franco, M; Kephart, G M; Gleich, G J

1998-07-01

Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Acute oral administration of low doses of methylphenidate targets calretinin neurons in the rat septal area

PubMed Central

García-Avilés, Álvaro; Albert-Gascó, Héctor; Arnal-Vicente, Isabel; Elhajj, Ebtisam; Sanjuan-Arias, Julio; Sanchez-Perez, Ana María; Olucha-Bordonau, Francisco

2015-01-01

Methylphenidate (MPD) is a commonly administered drug to treat children suffering from attention deficit hyperactivity disorder (ADHD). Alterations in septal driven hippocampal theta rhythm may underlie attention deficits observed in these patients. Amongst others, the septo-hippocampal connections have long been acknowledged to be important in preserving hippocampal function. Thus, we wanted to ascertain if MPD administration, which improves attention in patients, could affect septal areas connecting with hippocampus. We used low and orally administered MPD doses (1.3, 2.7 and 5 mg/Kg) to rats what mimics the dosage range in humans. In our model, we observed no effect when using 1.3 mg/Kg MPD; whereas 2.7 and 5 mg/Kg induced a significant increase in c-fos expression specifically in the medial septum (MS), an area intimately connected to the hippocampus. We analyzed dopaminergic areas such as nucleus accumbens and striatum, and found that only 5 mg/Kg induced c-fos levels increase. In these areas tyrosine hydroxylase correlated well with c-fos staining, whereas in the MS the sparse tyrosine hydroxylase fibers did not overlap with c-fos positive neurons. Double immunofluorescence of c-fos with neuronal markers in the septal area revealed that co-localization with choline acethyl transferase, parvalbumin, and calbindin with c-fos did not change with MPD treatment; whereas, calretinin and c-fos double labeled neurons increased after MPD administration. Altogether, these results suggest that low and acute doses of methylphenidate primary target specific populations of caltretinin medial septal neurons. PMID:25852493

Boric Acid Reduces the Formation of DNA Double Strand Breaks and Accelerates Wound Healing Process.

PubMed

Tepedelen, Burcu Erbaykent; Soya, Elif; Korkmaz, Mehmet

2016-12-01

Boron is absorbed by the digestive and respiratory system, and it was considered that it is converted to boric acid (BA), which was distributed to all tissues above 90 %. The biochemical essentiality of boron element is caused by boric acid because it affects the activity of several enzymes involved in the metabolism. DNA damage repair mechanisms and oxidative stress regulation is quite important in the transition stage from normal to cancerous cells; thus, this study was conducted to investigate the protective effect of boric acid on DNA damage and wound healing in human epithelial cell line. For this purpose, the amount of DNA damage occurred with irinotecan (CPT-11), etoposide (ETP), doxorubicin (Doxo), and H 2 O 2 was determined by immunofluorescence through phosphorylation of H2AX (Ser139) and pATM (Ser1981) in the absence and presence of BA. Moreover, the effect of BA on wound healing has been investigated in epithelial cells treated with these agents. Our results demonstrated that H2AX (Ser139) foci numbers were significantly decreased in the presence of BA while wound healing was accelerated by BA compared to that in the control and only drug-treated cells. Eventually, the results indicate that BA reduced the formation of DNA double strand breaks caused by agents as well as improving the wound healing process. Therefore, we suggest that boric acid has important therapeutical effectiveness and may be used in the treatment of inflammatory diseases where oxidative stress and wound healing process plays an important role.

Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

PubMed

Hu, Jianpeng; Xuan, Xujun; Han, Conghui; Hao, Lin; Zhang, Peiying; Chen, Meng; He, Houguang; Fan, Tao; Dong, Binzheng

2012-03-01

To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

Serum YKL-40 as a marker of liver fibrosis in patients with non-alcoholic fatty liver disease.

PubMed

Kumagai, Erina; Mano, Yohei; Yoshio, Sachiyo; Shoji, Hirotaka; Sugiyama, Masaya; Korenaga, Masaaki; Ishida, Tsuyoshi; Arai, Taeang; Itokawa, Norio; Atsukawa, Masanori; Hyogo, Hideyuki; Chayama, Kazuaki; Ohashi, Tomohiko; Ito, Kiyoaki; Yoneda, Masashi; Kawaguchi, Takumi; Torimura, Takuji; Nozaki, Yuichi; Watanabe, Sumio; Mizokami, Masashi; Kanto, Tatsuya

2016-10-14

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. YKL-40, chitinase-like protein expressed in multiple tissues including liver, is involved in cell proliferation, inflammation and remodeling of the extracellular matrix. The aim of this study was to assess whether serum YKL-40 levels are associated with liver fibrosis in NAFLD patients. Serum YKL-40 levels were quantified in 111 NAFLD patients and 23 HCC patients with NAFLD. To identify the source of YKL-40, immunofluorescence staining of liver specimens from NAFLD patients was performed. Serum YKL-40 levels in NAFLD patients increased in accordance with the progression of liver fibrosis. Multivariate analysis revealed that YKL-40 was one of the independent factors significantly associated with severe fibrosis (F3-4). We established a new predictive model for fibrosis of NAFLD, using logistic regression analysis: YKL-40 based fibrosis score = -0.0545 + type IV collagen 7s * 0.3456 + YKL-40 * 0.0024. Serum YKL-40 levels of HCC patients with non-cirrhotic NAFLD were significantly higher than those without HCC. Immunofluorescence staining showed that YKL-40 was expressed by macrophages in liver tissue of NAFLD patients. In conclusion, macrophage-derived YKL-40 is a feasible biomarker of liver fibrosis in NAFLD patients.

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

Identification of various cell culture models for the study of Zika virus

PubMed Central

Himmelsbach, Kiyoshi; Hildt, Eberhard

2018-01-01

AIM To identify cell culture models supportive for Zika virus (ZIKV) replication. METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis. RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines. CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research. PMID:29468137

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

PubMed

Yin, Xiaotao; Li, Fanglong; Jin, Yipeng; Yin, Zhaoyang; Qi, Siyong; Wu, Shuai; Wang, Zicheng; Wang, Lin; Yu, Jiyun; Gao, Jiangping

2017-03-01

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.

PubMed

Park, Miey; Shin, Hae J; Lee, Soo Y; Ahn, Tae I

2005-01-01

Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.

Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

PubMed Central

Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

2016-01-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns.

PubMed

Szafran, Adam T; Mancini, Maureen G; Nickerson, Jeffrey A; Edwards, Dean P; Mancini, Michael A

2016-03-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. Copyright © 2015 Elsevier Inc. All rights reserved.

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed Central

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-01-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis. PMID:6361052

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

PubMed

Sampson, J S; Wilkinson, H W; Tsang, V C; Brake, B J

1983-12-01

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis.

Characterizations of double pulsing in neutron multiplicity and coincidence counting systems

DOE PAGES

Koehler, Katrina E.; Henzl, Vladimir; Croft, Stephen; ...

2016-06-29

Passive neutron coincidence/multiplicity counters are subject to non-ideal behavior, such as double pulsing and dead time. It has been shown in the past that double-pulsing exhibits a distinct signature in a Rossi-alpha distribution, which is not readily noticed using traditional Multiplicity Shift Register analysis. But, it has been assumed that the use of a pre-delay in shift register analysis removes any effects of double pulsing. Here, we use high-fidelity simulations accompanied by experimental measurements to study the effects of double pulsing on multiplicity rates. By exploiting the information from the double pulsing signature peak observable in the Rossi-alpha distribution, themore » double pulsing fraction can be determined. Algebraic correction factors for the multiplicity rates in terms of the double pulsing fraction have been developed. We also discuss the role of these corrections across a range of scenarios.« less

Identification and phylogenetic analysis of a sheep pox virus isolated from the Ningxia Hui Autonomous Region of China.

PubMed

Zhu, X L; Yang, F; Li, H X; Dou, Y X; Meng, X L; Li, H; Luo, X N; Cai, X P

2013-05-14

An outbreak of sheep pox was investigated in the Ningxia Hui Autonomous Region in China. Through immunofluorescence testing, isolated viruses, polymerase chain reaction identification, and electron microscopic examination, the isolated strain was identified as a sheep pox virus. The virus was identified through sequence and phylogenetic analysis of the P32 gene, open reading frame (ORF) 095, and ORF 103 genes. This study is the first to use the ORF 095 and ORF 103 genes as candidate genes for the analysis of sheep pox. The results showed that the ORF 095 and ORF 103 genes could be used for the genotyping of the sheep pox virus.

[Wood smoke condensate induced epithelial-mesenchymal transition in human airway epithelial cells].

PubMed

Li, Wenxi; Zou, Weifeng; Li, Bing; Ran, Pixin

2014-01-01

To observe the detrimental effects of wood smoke condensate (WSC) exposure on human bronchial epithelial cells (HBEC), and to explore the expression of epithelial-mesenchymal transition (EMT) markers in HBEC exposed to WSC. HBEC were exposed respectively to 5, 10, 20, 40 and 50 mg/L of WSC /CSC for 7 days, with control groups only in cell culture medium at the same time, then the total cytoactivity was detected by cell counting kit-8. After observing the cellular morphology of WSC-stimulated HBEC. Western blot and immunofluorescence method were used to evaluate the expression levels of type I collagen, vimentin, E-cad and MMP-9 in HBEC exposed to WSC (10 mg/L) and cigarette smoke condensate (CSC) (10 mg/L) for 7 days. Statistical evaluation of the continuous data was performed by ANOVA. Independent-Samples t-test for between-group comparisons. After 7 days of exposure to WSC, HBEC manifested a morphological characteristic of loss of cell-cell contact and elongated shape. The level of E-cad was decreased in WSC exposure groups (Western blot: 0.30 ± 0.05, F = 22.07, P < 0.05) compared with the groups without WSC exposure (Western blot: 0.59 ± 0.08, F = 22.07, P < 0.05). In contrast, an upregulation in expression of type I collagen (Western blot: 0.58 ± 0.04 vs 0.26 ± 0.02, F = 119.72, P < 0.05) and MMP-9 (0.56 ± 0.08 vs 0.19 ± 0.03, F = 21.79, P < 0.05) was observed in the presence of WSC, compared with the control groups. Immunofluorescence analysis showed that after a 7-day exposure to WSC in these cells, the E-cad protein was lost whereas type I collagen, vimentin and MMP-9 were acquired. Both Western blot and immunofluorescence analysis showed no difference in expression levels of E-cad, type I collagen, vimentin and MMP-9 between WSC and CSC exposure groups. WSC exposure could induce EMT-like process in human airway epithelial cells.

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

PubMed

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

Wavelength-resolved emission spectroscopy of the alkoxy and alkylthio radicals in a supersonic jet

NASA Technical Reports Server (NTRS)

Misra, Prabhakar; Zhu, Xinming; Hsueh, Ching-Yu; Kamal, Mohammed M.

1993-01-01

Wavelength-resolved emission spectra of methoxy (CH3O) and methylthio (CH3S) radicals have been obtained in a supersonic jet environment with a resolution of 0.3 nm by dispersing the total laser-induced fluorescence with a 0.6 m monochromator. A detailed analysis of the single vibronic level dispersed fluorescence spectra yields the following vibrational frequencies for CH3O in the X(2)E state; nu(sub 1 double prime) = 2953/cm, nu(sub 2 double prime) = 1375/cm, nu(sub 3 double prime) = 1062/cm, nu(sub 4 double prime) = 2869/cm, nu(sub 5 double prime) = 1528/cm and nu(sub 6 double prime) = 688/cm. A similar analysis of the wavelength-resolved emission spectra of CH3S provides the following ground state vibrational frequencies: nu(sub 2 double prime) = 1329/cm, nu(sub 3 double prime) = 739/cm and nu(sub 6 double prime) = 601/cm. An experimental uncertainty of 20/cm is estimated for the assigned frequencies.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

Double symbolic joint entropy in nonlinear dynamic complexity analysis

NASA Astrophysics Data System (ADS)

Yao, Wenpo; Wang, Jun

2017-07-01

Symbolizations, the base of symbolic dynamic analysis, are classified as global static and local dynamic approaches which are combined by joint entropy in our works for nonlinear dynamic complexity analysis. Two global static methods, symbolic transformations of Wessel N. symbolic entropy and base-scale entropy, and two local ones, namely symbolizations of permutation and differential entropy, constitute four double symbolic joint entropies that have accurate complexity detections in chaotic models, logistic and Henon map series. In nonlinear dynamical analysis of different kinds of heart rate variability, heartbeats of healthy young have higher complexity than those of the healthy elderly, and congestive heart failure (CHF) patients are lowest in heartbeats' joint entropy values. Each individual symbolic entropy is improved by double symbolic joint entropy among which the combination of base-scale and differential symbolizations have best complexity analysis. Test results prove that double symbolic joint entropy is feasible in nonlinear dynamic complexity analysis.

Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

DTIC Science & Technology

1982-06-18

areas !i). Presently, the only certain method of identification is through the use of rigidly standardized reference antiserum in a virus plaque...low passaged or unpassaged dengue virus from humans or insects using an indirect immunofluorescence 71 test. MATERIALS AND METHODS :, j Cell cultures...streptomycin. Maintanance medium for infected cell cultures consisted of the appropriate growth medium containing 0.4% bovine plasma albumin instead of FBS

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

NASA Astrophysics Data System (ADS)

Li, Longhui; Rao, Gong; Lv, Xiaohua; Chen, Ruixi; Cheng, Xiaofeng; Wang, Xiaojun; Zeng, Shaoqun; Liu, Xiuli

2018-02-01

Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.

Gemella sanguinis endocarditis with c-ANCA/anti-PR-3-associated immune complex necrotizing glomerulonephritis with a ‘full-house’ pattern on immunofluorescence microscopy

PubMed Central

Rousseau-Gagnon, Mathieu; Riopel, Julie; Desjardins, Anne; Garceau, Daniel; Agharazii, Mohsen; Desmeules, Simon

2013-01-01

A 67-year-old man was evaluated for haematuria, with a rising creatinine level from 88 to 906 µmol/L and positive c-anti-neutrophil cytoplasm antibody (ANCA)/anti-proteinase 3 (anti-PR3). A kidney biopsy revealed necrotizing glomerulonephritis with a ‘full-house’ pattern on immunofluorescence microscopy. Echocardiography and blood cultures growing Gemella sanguinis diagnosed endocarditis. Dialysis was required for a month. Three months later, following valve replacement, glucocorticoids and 2 months of antibiotic therapy, the creatinine level decreased to 62 µmol/L and c-ANCA/anti-PR3 disappeared. This first case of c-ANCA/anti-PR3 positive glomerulonephritis with a ‘full-house’ immunofluorescence pattern due to bacterial endocarditis underlines the importance of ruling out infection with ANCA positivity or kidney biopsy suggestive of lupus nephritis. PMID:26064489

An erythrodermic variant of pemphigus foliaceus with puzzling histologic and immunopathologic features.

PubMed

Peterson, Jennifer D; Worobec, Sophie M; Chan, Lawrence S

2007-01-01

Pemphigus foliaceus is an autoimmune blistering disorder that affects the skin owing to autoantibodies against desmoglein 1. We employed clinical, histologic, immunopathologic, and serum laboratory studies to investigate a case of an erythrodermic variant of pemphigus foliaceus in an elderly man following treatment with bisoprolol-hydrochlorothiazide. Early histopathology revealed psoriasiform dermatitis, but later biopsies showed subcorneal and granular layer separation with neutrophilic infiltrate. Direct immunofluorescence showed intercellular deposits of immunoglobulin G throughout the epidermis, granular staining of C3 along the basement membrane zone, and fibrin and C3 deposition around the blood vessels. Indirect immunofluorescence on monkey esophagus showed a titer of greater than 1:1,280. Indirect immunofluorescence on rat bladder, antinuclear antibody, lupus panel, and kidney function panel were all negative. There are no reports in the literature of pemphigus foliaceus being induced by bisoprolol, but reports exist of propanolol resulting in drug-induced pemphigus foliaceus.

Characterization of Bombyx mori nucleopolyhedrovirus Bm17.

PubMed

Shen, Hongxing; Wang, Rudu; Han, Qinggong; Zhang, Wen; Nin, Bin; Zhou, Yang; Shao, Shihe; Yao, Qin; Chen, Keping; Liu, Xiaoyong

2013-10-01

Open reading frame17 (Bm17) of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this report, we describe the characterization of Bm17. Reversed transcriptive-PCR (RT-PCR) and Western blot analysis demonstrated that Bm17 was expressed as a late gen. Immunofluorescence analysis by confocal microscopy showed that BM17 protein was localized on cytoplasm and nucleus of infected cells. These results show that BM17 was a late protein localized in cytoplasm and nucleus. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine].

PubMed

Yang, Du; Shuai, Yuan; Zhifei, Zhou; Lizheng, Wu; Lulu, Wang; Xing'an, Wu; Xiaojing, Wang

2017-04-01

To explore the effect of nicotine on the autophagy level of human periodontal ligament cells (hPDLCs). Periodontal tissues collected from premolars for orthodontic treatment reasons were used to culture hPDLCs. Western blot analysis was performed to test the most optimal time and concentration of nicotine on the autophagy level of the hPDLCs. Transmission electron microscope and immunofluorescence observation were carried out to detect the form of autophagosomes and expression of autophagy related protein LC3 in hPDLCs under this optimal condition. Protein expression of LC3Ⅱ was up regulated with the 12 h nicotine stimulating. Besides that, the up regulation of the protein expression of LC3Ⅱ was concentration dependent and nicotine with a concentration of 1×10⁻⁵ mol·L⁻¹ was the most optimal condition. Transmission electron microscope and immunofluorescence observations indicated that nicotine would activate the autophagy level of hPDLCs by increasing the number of autophagosomes and up regulating the expression of autophagy related protein LC3. Nicotine could increase autophagy level of hPDLCs, thus affecting the occurrence and development of smoking related periodontitis.

The minute virus of mice exploits different endocytic pathways for cellular uptake

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcin, Pierre O.; Panté, Nelly, E-mail: pante@zoology.ubc.ca

The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy andmore » flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. - Highlights: • MVMp uptake takes place at the leading edge of migrating cells. • MVMp exploits a variety of endocytic pathways. • MVMp could use clathrin- and caveolin-mediated endocytosis. • MVMp could also use clathrin-independent carriers for cellular uptake.« less

A new method for in vivo visualization of vessel remodeling using a near-infrared dye

PubMed Central

Billaud, Marie; Ross, Jeremy A; Greyson, Mark A; Bruce, Anthony C; Seaman, Scott A; Heberlein, Katherine R; Han, Jenny; Best, Angela K; Peirce, Shayn M; Isakson, Brant E

2011-01-01

Intro Vascular obstructive events can be partially compensated for by remodeling processes that increase vessel diameter and collateral tortuosity. However, methods for visualizing remodeling events in vivo and with temporal comparisons from the same animal remain elusive. Methods Using a novel infrared conjugated polyethylene glycol dye, we investigated the possibility of intravital vascular imaging of the mouse ear before and after ligation of the primary feeder artery. For comparison, we used two different mouse models known to have impaired vascular remodeling post ligation (i.e. aged and PAI-1−/− mice). The results obtained with the infrared dye were confirmed using immunofluorescence labeling of the ear microvasculature with confocal microscopy. Results After ligation, increases in vessel diameter (between 10% and 60%) and tortuosity (approximately 15%) were observed in C57Bl/6 mice using both the infrared dye and the immunofluorescence technique. However, aged C57Bl/6 and PAI-1−/− mice did not show vascular remodeling following ligation. Conclusion Vascular remodeling can be visualized and accurately quantified using a new infrared dye in vivo. This analysis technique could be generally employed for quantitative investigations of changes in vascular remodeling. PMID:21418375

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

PubMed Central

Dobrivojević, Marina; Bohaček, Ivan; Erjavec, Igor; Gorup, Dunja; Gajović, Srećko

2013-01-01

Aim To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Methods Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Results Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. Conclusion MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema. PMID:23444240

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents.

PubMed

Dobrivojević, Marina; Bohaček, Ivan; Erjavec, Igor; Gorup, Dunja; Gajović, Srećko

2013-02-01

To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema.

Ten cases of severe oral lichen planus showing granular C3 deposition in oral mucosal basement membrane zone.

PubMed

Hashimoto, Takashi; Fukuda, Aoi; Himejima, Akio; Morita, Shosuke; Tsuruta, Daisuke; Koga, Hiroshi; Krol, Rafal P; Ishii, Norito

2015-01-01

Oral lichen planus (OLP) may show depositions of immunoglobulins and complement components in oral mucosal basement membrane zone (BMZ) in direct immunofluorescence, although these finding are not frequently seen. We collected and examined ten cases of severe OLP showing granular C3 deposition in BMZ. In addition to clinical, histopathological and direct immunofluorescence assessments, we performed various immune-serological tests, including indirect immunofluorescence of normal human skin and 1M NaCl-split skin, immunoblotting of normal human epidermal and dermal extracts, recombinant proteins of BP180 NC16a and C-terminal domains, concentrated culture supernatant of HaCaT cells and purified human laminin-332, and enzyme-linked immunosorbent assays for BP230 and BP180. Direct immunofluorescence showed C3 deposition in BMZ exclusively of granular pattern in 7 cases and of both granular and linear patterns in 3 cases. The 10 cases showed no positive reactivity for either IgG or IgA antibodies in any immuno-serological tests. Detailed analyses of clinical, histopathological and immunological findings revealed striking female prevalence, although other parameters were in general characteristic of OLP. Granular C3 deposition in oral BMZ may be one of the characteristic features of severe OLP, although mechanisms for C3 deposition and its pathogenic role in OLP are currently unknown.

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy.

PubMed

Sun, Yulong; Ip, Philbert; Chakrabartty, Avijit

2017-09-03

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

NASA Astrophysics Data System (ADS)

Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui

2011-01-01

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.

Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma.

PubMed

Sato, A; Ochi, H; Harada, Y; Yogo, T; Kanno, N; Hara, Y

2017-01-01

The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of CD147 and matrix metalloproteinase-11 in colorectal cancer and their relationship to clinicopathological features.

PubMed

Tian, Xiuyun; Ye, Chunxiang; Yang, Yongyong; Guan, Xiaoya; Dong, Bin; Zhao, Min; Hao, Chunyi

2015-10-27

This study aimed to investigate the expression of CD147 and MMP-11 in human colorectal cancer (CRC) and to evaluate their clinical significance. Real-time polymerase chain reaction was used to evaluate CD147 and MMP-11 mRNA level in 56 pairs of fresh CRC samples matched with adjacent normal mucosa. The protein expression of CD147 and MMP-11 in CRC specimens and corresponding normal colorectal mucosa were evaluated by immunohistochemistry on CRC tissue microarrays. Expression and co-localization of these two proteins in human colorectal cancer tissue were also evaluated by laser scanning confocal microscopy. Furthermore, their correlations with clinicopathological factors and overall survival after surgery were evaluated. Both CD147 and MMP-11 were demonstrated to be over-expressed at mRNA level (P < 0.001, both) and protein level (P < 0.001, both) in CRC tissue than paired normal mucosa. Spearman rank test showed a positive correlation between these two proteins (P = 0.025). Immunofluorescence double staining confirmed the co-localization of CD147 and MMP-11 in paraffin-embedded tissues of CRC patients. Expression of CD147 and MMP-11 were both correlated with CRC lymph node metastasis (P = 0.021 and P = 0.031, respectively), distant metastasis (P < 0.001 and P = 0.013, respectively) and TNM stage (P = 0.006 and P = 0.049, respectively). Univariate survival analysis showed that both CD147 and MMP11 expression was significantly associated with shorter survival time (P = 0.001 and P = 0.009, respectively). Additionally, in multivariate analysis, both CD147 and MMP-11 were proved to be independent prognostic factors (P = 0.009, 0.028, respectively). These results indicated that both CD147 and MMP-11 may be involved in the progression of colorectal cancer, and they are potential prognostic factors and might become new therapeutic targets for CRC patients.

Abundance of the Organic Anion-transporting Polypeptide OATP4A1 in Early-Stage Colorectal Cancer Patients: Association With Disease Relapse.

PubMed

Buxhofer-Ausch, Veronika; Sheikh, Maidah; Ausch, Christoph; Zotter, Simone; Bauer, Heike; Mollik, Marina; Reiner, Angelika; Gleiss, Andreas; Jäger, Walter; Sebesta, Christian; Kriwanek, Stephan; Thalhammer, Theresia

2018-05-03

The abundance of OATP4A1 in colorectal cancer (CRC) might be related to tumor progression. This was studied by immunohistochemistry on paraffin-embedded samples obtained from 178 patients (43 patients with a relapse within 5 y) with early-stage CRC. Positivity for OATP4A1 in tumor cells and noncancerous mucosal cells was proved by double-immunofluorescence staining with antibodies against OATP4A1 and keratin 8, whereas antibodies against appropriate CD markers were used to identify immune cells. Automated microscopic image analysis was used to measure the percentage of OATP4A1-positive cells and OATP4A1 staining intensity in tumor, immune, and adjacent normal-looking mucosal cells separately, as well as in the mucosal and immune cells of 14 nonmalignant tissue samples. In CRC the percentage of OATP4A1-positive cells, but not staining intensity, was significantly higher in tumor and mucosal cells adjacent to the tumor compared to the mucosa of nonmalignant samples (P<0.001 each). No difference was registered between immune cells in malignant and nonmalignant samples. Importantly, high levels of OATP4A1 in immune (odds ratio, 0.73; confidence interval, 0.63-0.85; P<0.001), and tumor cells (odds ratio, 0.79; confidence interval, 0.69-0.91; P<0.001) are significantly associated with a low risk of recurrence and also significantly enhance the discriminative power of other clinical parameters [such as International Union Against Cancer (UICC), adjuvant therapy, localization of the primary tumor] of the risk of relapse (receiver operating characteristics analysis; P=0.002). Using an advanced digital microscopic quantification procedure, we showed that OATP4A1 abundance is negatively associated with tumor recurrence in early-stage CRC. This digital scoring procedure may serve as a novel tool for the assessment of potential prognostic markers in early-stage CRC.

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells.

PubMed

Immenschuh, S; Tan, M; Ramadori, G

1999-01-01

Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the body's tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide. Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols. Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner. This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor N(G)-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells. The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.

Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

PubMed

Senesi, Pamela; Luzi, Livio; Montesano, Anna; Mazzocchi, Nausicaa; Terruzzi, Ileana

2013-07-19

Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. We performed a dose-response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

Identification and characterization of functional centromeres of the common bean.

PubMed

Iwata, Aiko; Tek, Ahmet L; Richard, Manon M S; Abernathy, Brian; Fonsêca, Artur; Schmutz, Jeremy; Chen, Nicolas W G; Thareau, Vincent; Magdelenat, Ghislaine; Li, Yupeng; Murata, Minoru; Pedrosa-Harand, Andrea; Geffroy, Valérie; Nagaki, Kiyotaka; Jackson, Scott A

2013-10-01

In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Surveillance of Hantaviruses in Poland: A Study of Animal Reservoirs and Human Hantavirus Disease in Subcarpathia

PubMed Central

Niemcewicz, Marcin; Bielawska-Drózd, Agata; Nowakowska, Anna; Gaweł, Jerzy; Pitucha, Grzegorz; Joniec, Justyna; Zielonka, Katarzyna; Marciniak-Niemcewicz, Anna; Kocik, Janusz

2014-01-01

Abstract The first cluster of hemorrhagic fever with renal syndrome (HFRS) in Poland was identified in 2007 in the Subcarpathian region. The natural environment of this area is a key habitat for hantavirus vectors. The animal reservoir of existing human HFRS clusters was studied to assess the occurrence of viruses (including Tula virus, Puumala virus, and Dobrava–Belgrade virus) among rodents. We examined 70 suspected human cases with symptoms corresponding to the clinical picture of HFRS. Serological analysis (indirect immunofluorescence assay and immunoblot) confirmed the presence of anti-hantavirus antibodies in 18 patients, which were surveyed with regard to developed symptoms and presumed rodent contact. Seroepidemiological analysis of newly confirmed human cases was performed, putative areas of human exposure were studied, and 194 rodents were subsequently captured from identified areas. Internal organs (lungs, heart, spleen, bladder, and kidneys) were collected from 64 Apodemus flavicollis, 55 Apodemus agrarius, 40 Myodes glareolus, 21 Mus musculus, and 14 Microtus arvalis and tested for the presence of hantavirus RNA by reverse transcription and subsequent real-time PCR. Positive samples were also tested by indirect immunofluorescence. Animal reservoir surveillance enabled the first detection of Puumala virus and Dobrava–Belgrade virus among animals in Poland. Furthermore, some places where rodents were captured correlated with areas of residence of laboratory-confirmed human cases and likely detected virus species. Moreover, three species of hantaviruses coexisting in a relatively small area were identified. PMID:24902039

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

PubMed

Gorrell, M D; Wickson, J; McCaughan, G W

1991-04-15

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.

Mouse hepatitis virus immunofluorescence in formalin- or Bouin's-fixed tissues using trypsin digestion.

PubMed

Brownstein, D G; Barthold, S W

1982-02-01

Mouse hepatitis viral antigens were demonstrated by immunofluorescence in formalin- and Bouin's-fixed tissues processed routinely for histopathology followed by partial digestion with trypsin. Staining was superior in tissues fixed in formalin and was not diminished in tissue sections from paraffin blocks stored at room temperature as long as 2 years. The relative ease of this procedure and the commercial availability of reagents makes this a useful technique for the definitive diagnosis of mouse hepatitis virus infection.

The importance of direct immunofluorescence in pemphigus herpetiformis diagnosis*

PubMed Central

de Faria, Paula Carolina Pessanha; Cruz, Camila Caberlon; Abulafia, Luna Azulay; Maceira, Juan Manuel Pineiro; Cassia, Flávia de Freire; Medeiros, Paula Mota

2017-01-01

Pemphigus herpetiformis is an autoimmune bullous disease, that combines clinical features of dermatitis herpetiformis and linear IgA bullous dermatosis and immunological characteristics of pemphigus, which makes this disease peculiar and this diagnosis rarely suspected in the first evaluation of the patient. The reported case is of a patient with clinically bullous disease similar to dermatitis herpetiformis, whose multiple biopsies were inconclusive, and only after direct immunofluorescence with a pemphigus pattern (intraepidermal intercellular pattern) the confirmation of the diagnosis was possible. PMID:29267475

Bacillus Anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but not to Macrophages

DTIC Science & Technology

2007-09-01

immunofluorescence (IFM) and light microscopy. Samples were fixed in forma- lin, stained with immunofluorescent dyes (as described below) or spore stain ( malachite ...BEC. Adherence was assessed by microscopic observation of the infected cells stained with malachite green and counterstaining of the BEC. For enzymatic...this significant difference, BEC infected with spores were stained with malachite green and counter- stained with Wright-Giemsa (Fig. 1B and C). This

Single Versus Double Lung Retransplantation Does Not Affect Survival Based on Previous Transplant Type.

PubMed

Schumer, Erin M; Rice, Jonathan D; Kistler, Amanda M; Trivedi, Jaimin R; Black, Matthew C; Bousamra, Michael; van Berkel, Victor

2017-01-01

Survival following retransplantation with a single lung is worse than after double lung transplant. We sought to characterize survival of patients who underwent lung retransplantation based on the type of their initial transplant, single or double. The United Network for Organ Sharing database was queried for adult patients who underwent lung retransplantation from 2005 onward. Patients were excluded if they underwent more than one retransplantation. The patient population was divided into 4 groups based on first followed by second transplant type, respectively: single then single, double then single, double then double, and single then double. Descriptive analysis and Kaplan-Meier survival analysis were performed. A p value less than 0.05 was considered significant. A total of 410 patients underwent retransplantation in the study time period. Overall mean survival for all patients who underwent retransplantation was 1,213 days. Kaplan-Meier survival analysis demonstrated no difference in graft survival between the 4 study groups (p = 0.146). There was no significant difference in graft survival between recipients of retransplant with single or double lungs when stratified by previous transplant type. These results suggest that when retransplantation is performed, single lung retransplantation should be considered, regardless of previous transplant type, in an effort to maximize organ resources. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

PubMed Central

Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

2012-01-01

Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

The impact of the IGF-1 system of cancer cells on radiation response - An in vitro study.

PubMed

Venkatachalam, Senthiladipan; Mettler, Esther; Fottner, Christian; Miederer, Matthias; Kaina, Bernd; Weber, Matthias M

2017-12-01

Overexpression of the insulin-like growth factor-1 receptor (IGF-1R) is associated with increased cell proliferation, differentiation, transformation, and tumorigenicity. Additionally, signaling involved in the resistance of cancer cells to radiotherapy originates from IGF-1R. The purpose of this study was to investigate the role of the IGF-1 system in the radiation response and further evaluate its effect on the expression of DNA repair pathway genes. To inhibit the IGF-1 system, we stably transfected the Caco-2 cell line to express a kinase-deficient IGF-1R mutant. We then studied the effects of this mutation on cell growth, the response to radiation, and clonogenic survival, as well as using a cell viability assay to examine DNA damage and repair. Finally, we performed immunofluorescence for γ-H2AX to examine double-strand DNA breaks and evaluated the expression of 84 key genes involved in DNA repair with a real-time PCR array. Mutant IGF-1R cells exhibited significantly blunted cell growth and viability, compared to wild-type cells, as well as reduced clonogenic survival after γ-irradiation. However, mutant IGF-1R cells did not show any significant delays in the repair of radiation-induced DNA double-strand breaks. Furthermore, expression of mutant IGF-1R significantly down-regulated the mRNA levels of BRCA2, a major protein involved in homologous recombination DNA repair. These results indicate that blocking the IGF-1R-mediated signaling cascade, through the expression of a kinase-deficient IGF-1R mutant, reduces cell growth and sensitizes cancer cells to ionizing radiation. Therefore, the IGF-1R system could be a potential target to enhance radio-sensitivity and the efficacy of cancer treatments.

A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle--dependent changes in mast cell--nerve fiber contacts in murine skin.

PubMed

Botchkarev, V A; Eichmüller, S; Peters, E M; Pietsch, P; Johansson, O; Maurer, M; Paus, R

1997-04-01

Close contacts between mast cells (MC) and nerve fibers have previously been demonstrated in normal and inflamed skin by light and electron microscopy. A key step for any study in MC-nerve interactions in situ is to simultaneously visualize both communication partners, preferably with the option of double labelling the nerve fibers. For this purpose, we developed the following triple-staining technique. After paraformaldehyde-picric acid perfusion fixation, cryostat sections of back skin from C57BL/6 mice were incubated with a primary rat monoclonal antibody to substance P (SP), followed by incubation with a secondary goat-anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then applied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG. MCs were visualized by incubation with AMCA-labelled avidin, or (for a more convenient quantification of close MC-nerve fiber contacts) with a mixture of TRITC- and FITC-labelled avidins. Using this simple, novel covisualization method, we were able to show that MC-nerve associations in mouse skin are, contrary to previous suggestions, highly selective for nerve fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MCs preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR double-labelled nerve fibers. Compared with telogen values, there was a significant increase in the number of close contacts between MCs and tyrosine hydroxylase-IR fibers during late anagen, and between MCs and peptide histidine-methionine-IR and choline acetyl transferase-IR fibers during catagen.

In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

PubMed Central

Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

2009-01-01

BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen.

PubMed

Liu, Tiancheng; Wu, Lisa Y; Choi, Joseph K; Berkman, Clifford E

2009-05-01

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. (c) 2009 Wiley-Liss, Inc.

Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

PubMed Central

Müller, Brigitte; Ellinwood, N. M.; Lorenz, Birgit; Stieger, Knut

2018-01-01

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted. PMID:29765300

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Distinct signaling pathways leading to the induction of human β-defensin 2 by stimulating an electrolyticaly-generated acid functional water and double strand RNA in oral epithelial cells.

PubMed

Gojoubori, Takahiro; Nishio, Yukina; Asano, Masatake; Nishida, Tetsuya; Komiyama, Kazuo; Ito, Koichi

2014-04-01

Defensins, a major family of cationic antimicrobial peptides, play important roles in innate immunity. In the present study, we investigated whether double-stranded RNA (dsRNA), a by-product of RNA virus replication, can induce human β-defensins-2 (hBD-2) expression in oral epithelial cells (OECs). We also examined the hBD-2-inducible activity of acid-electrolyzed functional water (FW). The results indicated that both dsRNA- and FW-induced hBD-2 expression in OECs. The induction efficiency was much higher for FW than for dsRNA. FW-induced production of hBD-2 was clearly observed by immunofluorescence staining. A luciferase assay was performed with 1.2 kb of the 5'-untranslated region (5'-UTR) of the hBD-2 gene. The results indicated that the nuclear factor-kappa B (NF-κB)-binding site proximal to the translation initiation site was indispensable for dsRNA-stimulated hBD-2 expression, but not in the case of FW. Moreover, FW-stimulated hBD-2 expression did not depend on NF-κB activity; instead, FW inhibited NF-κB activity. Pretreatment of the cells with specific inhibitors against NF-κB further confirmed NF-κB-independent hBD-2 induction by FW. In analogy to the results for intestinal epithelial cells (IECs), the dsRNA signal, but not FW, was sensed by toll-like receptor 3 (TLR3) in OECs. These results suggested that hBD-2 expression induced by dsRNA and FW is regulated by distinct mechanisms in OECs.

Biphasic activation of nuclear factor-κB and expression of p65 and c-Rel following traumatic neuronal injury.

PubMed

Zhang, Huasheng; Zhang, Dingding; Li, Hua; Yan, Huiying; Zhang, Zihuan; Zhou, Chenhui; Chen, Qiang; Ye, Zhennan; Hang, Chunhua

2018-06-01

The transcription factor nuclear factor-κB (NF-κB) has been shown to function as a key regulator of cell death or survival in neuronal cells. Previous studies indicate that the biphasic activation of NF-κB occurs following experimental neonatal hypoxia-ischemia and subarachnoid hemorrhage. However, the comprehensive understanding of NF-κB activity following traumatic brain injury (TBI) is incomplete. In the current study, an in vitro model of TBI was designed to investigate the NF-κB activity and expression of p65 and c-Rel subunits following traumatic neuronal injury. Primary cultured neurons were assigned to control and transected groups. NF-κB activity was detected by electrophoretic mobility shift assay. Western blotting and immunofluorescence were used to investigate the expression and distribution of p65 and c-Rel. Reverse transcription-quantitative polymerase chain reaction was performed to assess the downstream genes of NF-κB. Lactate dehydrogenase (LDH) quantification and trypan blue staining were used to estimate the neuronal injury. Double peaks of elevated NF-κB activity were observed at 1 and 24 h following transection. The expression levels of downstream genes exhibited similar changes. The protein levels of p65 also presented double peaks while c-Rel was elevated significantly in the late stage. The results of the trypan blue staining and LDH leakage assays indicated there was no sustained neuronal injury during the late peak of NF-κB activity. In conclusion, biphasic activation of NF-κB is induced following experimental traumatic neuronal injury. The elevation of p65 and c-Rel levels at different time periods suggests that within a single neuron, NF-κB may participate in different pathophysiological processes.

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus

PubMed Central

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-01-01

Abstract DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure. PMID:29800455

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

PubMed

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

Expression of forkhead box transcription factor genes Foxp1 and Foxp2 during jaw development.

PubMed

Cesario, Jeffry M; Almaidhan, Asma A; Jeong, Juhee

2016-03-01

Development of the face is regulated by a large number of genes that are expressed in temporally and spatially specific patterns. While significant progress has been made on characterizing the genes that operate in the oral region of the face, those regulating development of the aboral (lateral) region remain largely unknown. Recently, we discovered that transcription factors LIM homeobox (LHX) 6 and LHX8, which are key regulators of oral development, repressed the expression of the genes encoding forkhead box transcription factors, Foxp1 and Foxp2, in the oral region. To gain insights into the potential role of the Foxp genes in region-specific development of the face, we examined their expression patterns in the first pharyngeal arch (primordium for the jaw) of mouse embryos at a high spatial and temporal resolution. Foxp1 and Foxp2 were preferentially expressed in the aboral and posterior parts of the first pharyngeal arch, including the developing temporomandibular joint. Through double immunofluorescence and double fluorescent RNA in situ hybridization, we found that Foxp1 was expressed in the progenitor cells for the muscle, bone, and connective tissue. Foxp2 was expressed in subsets of bone and connective tissue progenitors but not in the myoblasts. Neither gene was expressed in the dental mesenchyme nor in the oral half of the palatal shelf undergoing extensive growth and morphogenesis. Together, we demonstrated for the first time that Foxp1 and Foxp2 are expressed during craniofacial development. Our data suggest that the Foxp genes may regulate development of the aboral and posterior regions of the jaw. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

PubMed Central

Villarreal, Ashley M.; Adamson, Steven W.; Browning, Rebecca E.; Khem Raj, B.C.; Sajid, Muhammad Sohail; Karim, Shahid

2013-01-01

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and A. americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases. PMID:23499931

GnRH neurons of young and aged female rhesus monkeys co-express GPER but are unaffected by long-term hormone replacement

PubMed Central

Naugle, Michelle M.; Gore, Andrea C.

2014-01-01

Menopause is caused by changes in the function of the hypothalamic-pituitary-gonadal (HPG) axis that controls reproduction. Hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus orchestrate the activity of this axis and are regulated by hormonal feedback loops. The mechanisms by which GnRH responses to the primary regulatory sex-steroid hormone, estradiol (E2) are still poorly understood in the context of menopause. Our goal was to determine whether the G protein-coupled estrogen receptor (GPER) is co-expressed in adult primate GnRH neurons, and whether this changes with aging and/or E2 treatment. We used immunofluorescence double labeling to characterize the co-expression of GPER in GnRH perikarya and terminals in the hypothalamus. Young and aged rhesus macaques were ovariectomized and given long-term (~2 year) hormone treatments (E2, E2 + progesterone, or vehicle) selected to mimic currently prescribed hormone replacement therapies used for the alleviation of menopausal symptoms in women. We found that about half of GnRH perikarya co-expressed GPER, while only about 12% of GnRH processes and terminals in the median eminence (ME) were double labeled. Additionally, many GPER labeled processes were in direct contact with GnRH neurons, often wrapped around the perikarya and processes and in close proximity in the ME. These results extend prior work by showing robust colocalization of GPER in GnRH in a clinically relevant model, and support the possibility that GPER-mediated E2 regulation of GnRH occurs both in the soma and terminals in nonhuman primates. PMID:25428637

KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain.

PubMed

Rhodes, Kenneth J; Carroll, Karen I; Sung, M Amy; Doliveira, Lisa C; Monaghan, Michael M; Burke, Sharon L; Strassle, Brian W; Buchwalder, Lynn; Menegola, Milena; Cao, Jie; An, W Frank; Trimmer, James S

2004-09-08

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Hydrocortisone activation of human herpesvirus 8 viral DNA replication and gene expression in vitro.

PubMed

Hudnall, S D; Rady, P L; Tyring, S K; Fish, J C

1999-03-15

Patients undergoing chronic steroid therapy for organ transplantation are at increased risk for development of human herpes virus 8(HHV-8)-associated Kaposi's sarcoma (KS). It has also been reported that following steroid withdrawal, KS lesions often undergo partial or complete regression. We have examined the effect of corticosteroid treatment on HHV-8 replication, gene expression, and lytic protein expression in BCBL-1 cells in vitro. BCBL-1 cells were collected after culture for 24-72 hr with hydrocortisone (HC) 1-5 microM, phorbol ester 20 ng/ml (positive control), and culture medium only (negative control). HHV-8 genomic conformation was examined by Gardella gel analysis. mRNA expression of viral cyclin (v-Cyc), viral Bcl-2 (v-Bcl-2), viral macrophage inflammatory protein-I (v-MIP-I), viral interferon regulatory factor-1(v-IRF-1), and viral tegument protein (TP) was examined by RT-PCR Southern blot. Viral protein expression within the cells was examined by indirect immunofluorescence using 5 different HHV-8 positive antisera from 4 renal transplant recipients and 1 patient with classic KS. Gardella gel analysis revealed that HC induced an accumulation of the linear replicative genomic form of the virus in a time-dependent fashion. Southern blot analysis of the RT-PCR products revealed that HC induced increased expression of v-IRF-1, v-Bcl-2, and TP mRNA, with little discernible effect on v-Cyc, and v-MIP-I. Immunofluorescence revealed that HC induced increased numbers of cells expressing lytic antigens. These data indicate that hydrocortisone acts directly on BCBL-1 cells to activate the lytic cycle of HHV-8 and provide further support for the hypothesis that HHV-8 is activated in corticosteroid-treated immunocompromised patients.

Effects of an intravitreal injection of interleukin-35-expressing plasmid on pro-inflammatory and anti-inflammatory cytokines.

PubMed

Hou, Chao; Wu, Qianni; Ouyang, Chen; Huang, Ting

2016-09-01

In order to explore the potential effects of interleukin (IL)-35 on IL-10, transforming growth factor-β (TGF-β), interferon-γ (INF)-γ, IL-12 and IL-17, a pcDNA3.1‑IL-35 plasmid was injected into the vitreous cavity of BALB/c mice. Enzyme-linked immunosorbent assay, western blot analysis and quantitative PCR analysis were performed to confirm the successful expression of IL-35. Slit-lamp biomicroscopy, hematoxylin and eosin staining and immunofluorescence were employed to detect the status of eyes, and western blot analysis was performed to examine the expression of corneal graft rejection-related cytokines. There were no abnormalities in the eyes pre-mydriasis or post-mydriasis and no injuries to the cornea or retina following the injection of IL-35-expressing plasmid. An immunofluorescence assay detected the positive expression of IL-35 in corneal epithelial cells from IL-35‑injected mice and negative staining in the control group. Further study revealed that IL-35 enhanced the expression of IL-10 and TGF-β which reached their highest levels at 1 and 2 weeks after injection, respectively (p<0.01). Moreover, the expression of INF-γ and IL-12 was decreased significantly at 2 weeks after the injection of IL-35-expressing plasmid (p<0.05), and the expression of IL-17 was suppressed notably at 4 weeks after the injection (p<0.05). The intravitreal injection of IL-35-expressing plasmid in mice downregulates the expression of pro-inflammatory cytokines and upregulates the expression of anti-inflammatory cytokines. Thus, IL-35 may further be assessed as a potential target for the treatment of corneal graft rejection.

Low diagnostic and predictive value of anti-dsDNA antibodies in unselected patients with recent onset of rheumatic symptoms: results from a long-term follow-up Scandinavian multicentre study.

PubMed

Compagno, M; Jacobsen, S; Rekvig, O P; Truedsson, L; Heegaard, N H; Nossent, J; Jönsen, A; Jacobsen, R S; Eilertsen, G Ø; Sturfelt, G; Bengtsson, A A

2013-01-01

To verify the diagnostic accuracy of anti-double-stranded DNA (anti-dsDNA) antibodies detected by the Crithidia luciliae immunofluorescence test (CLIFT) in a cohort of unselected patients, referred to a rheumatologist due to recent onset of rheumatic symptoms. A total of 1073 consecutive patients were screened for anti-nuclear antibodies (ANAs). Serum samples from 292 ANA-positive and 292 matching ANA-negative patients were tested three times for anti-dsDNA antibodies, using two different CLIFT kits (ImmunoConcepts(®) and Euroimmun(®)). An initial clinical diagnosis was made by rheumatologists unaware of the results. The diagnoses were updated after a median follow-up of 4.8 years. CLIFT was positive at least once in 60 patients but only 23 patients were CLIFT positive in all of the assays. Diagnosis of systemic lupus erythematosus (SLE) was made initially in 65 patients, of whom 24 (37%) were CLIFT positive. Many other diagnoses were observed among the CLIFT-positive patients. Overall, 16 (5.5%) ANA-negative patients were CLIFT positive. After approximately 5 years, the diagnosis of SLE remained unchanged in 63 patients (23 CLIFT positive) and altered in only two (one CLIFT positive). Among the 36 CLIFT-positive patients who were not diagnosed with SLE at study entry, only one developed SLE during the follow-up period. CLIFT was not reliable as a diagnostic tool in unselected patients with rheumatic symptoms. ANAs were of little value as a screening test before the CLIFT analysis. CLIFT had surprisingly low positive predictive value (PPV) for the diagnosis of SLE despite its high specificity. For non-SLE patients, being CLIFT positive poses little risk of developing SLE within 5 years.

C-reactive protein expression is up-regulated in apical lesions of endodontic origin in association with interleukin-6.

PubMed

Garrido, Mauricio; Dezerega, Andrea; Bordagaray, María José; Reyes, Montserrat; Vernal, Rolando; Melgar-Rodríguez, Samantha; Ciuchi, Pía; Paredes, Rodolfo; García-Sesnich, Jocelyn; Ahumada-Montalva, Pablo; Hernández, Marcela

2015-04-01

C-reactive protein (CRP) is the prototype component of acute-phase proteins induced ultimately by interleukin (IL)-6 in the liver, but it is unknown whether periradicular tissues locally express CRP. The present study aimed to identify whether CRP messenger RNA synthesis occurs in situ within apical lesions of endodontic origin (ALEOs) and healthy periodontal ligament and its association with IL-6 and to determine their protein levels and tissue localization. Patients with asymptomatic apical periodontitis and healthy volunteers presenting at the School of Dentistry, University of Chile, Santiago, Chile, were enrolled. ALEOs and healthy teeth were obtained and processed for either immunohistochemistry and double immunofluorescence to assess IL-6 and CRP tissue localization, whereas healthy periodontal ligaments were processed as controls for real-time reverse-transcription polymerase chain reaction for their RNA expression levels and multiplex assay to determine their protein levels. Statistic analysis was performed using the unpaired t test or Mann-Whitney test according to data distribution and Pearson correlation. IL-6 and CRP were synthesized in ALEOs, whereas their RNA expression and protein levels were significantly higher when compared with healthy periodontal ligament. IL-6 and CRP immunolocalized to the inflammatory cells, vascular endothelial cells, and mesenchymal cells. Both, IL-6 and CRP colocalized in ALEOs, and a positive correlation was found between their expression levels (P < .05). IL-6 and CRP messenger RNA are constitutively expressed in periodontal ligament and up-regulated in ALEOs along with higher protein levels. Given their pleiotropic effects, IL-6 and CRP protein levels in apical tissues might partially explain the development and progression of ALEOs as well as potentially asymptomatic apical periodontitis-associated systemic low-grade inflammation. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Expression of cholecystokinin2-receptor in rat and human L cells and the stimulation of glucagon-like peptide-1 secretion by gastrin treatment.

PubMed

Cao, Yang; Cao, Xun; Liu, Xiao-Min

2015-03-01

Gastrin is a gastrointestinal hormone secreted by G cells. Hypergastrinemia can improve blood glucose and glycosylated hemoglobin levels. These positive effects are primarily due to the trophic effects of gastrin on β-cells. In recent years, many receptors that regulate secretion of glucagon-like peptide 1 (GLP-1) have been identified in enteroendocrine L cell lines. This led us to hypothesize that, in addition to the trophic effects of gastrin on β-cells, L cells also express cholecystokinin2-receptor (CCK2R), which may regulate GLP-1 secretion and have synergistic effects on glucose homeostasis. Our research provides a preliminary analysis of CCK2R expression and the stimulating effect of gastrin treatment on GLP-1 secretion in a human endocrine L cell line, using RT-PCR, Western blot, immunocytochemistry, and ELISA analyses. The expression of proglucagon and prohormone convertase 3, which regulate GLP-1 biosynthesis, were also analyzed by real-time PCR. Double immunofluorescence labeling was utilized to assess the intracellular localization of CCK2R and GLP-1 in L cells harvested from rat colon tissue. Our results showed that CCK2R was expressed in both the human L cell line and the rat L cells. We also showed that treatment with gastrin, a CCK2R agonist, stimulated the secretion of GLP-1, and that this effect was likely due to increased expression of proglucagon and PCSK1 (also known as prohormone convertase 3 (PC3 gene)). These results not only provide a basis for the role gastrin may play in intestinal L cells, and may also provide the basis for the development of a method of gastrin-mediated glycemic regulation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Modulating the internalization of bacille Calmette-Guérin by cathelicidin in bladder cancer cells.

PubMed

Choi, Se Young; Kim, Soon-Ja; Chi, Byung Hoon; Kwon, Jong Kyou; Chang, In Ho

2015-04-01

To confirm the role of cathelicidin (LL-37) in the internalization of bacille Calmette-Guérin (BCG) into bladder cancer cells. Enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction analysis evaluated the changes in protein and messenger ribonucleic acid (RNA) expression with BCG incubation after LL-37 pretreatment in 5637 and T24 human bladder cancer cells. The internalization rate was evaluated by a double immunofluorescence assay, and confocal microscopy confirmed the function of LL-37 in BCG internalization. We also investigated the difference in internalization rates and cell viability between LL-37, anti-LL-37 antibody, and LL-37 plus anti-LL-37 antibody. The levels of LL-37 increased after BCG exposure in bladder cancer cells in dose- and time-dependent manners. Increasing LL-37 levels using recombinant LL-37 protein further dose dependently decreased BCG internalization in both cell lines. The internalization rates of BCG after LL-37 instillation were lower compared with the controls, and the internalization rate of BCG after anti-LL-37 antibody instillation was significantly higher compared with the controls in both cell lines (P <.05). Viability of LL-37 plus BCG group was higher compared with the BCG-alone group. The anti-LL-37 antibody plus BCG group had decreased cell viability compared with the BCG-alone group in both cell lines. Bladder cancer cells produce cathelicidin when infected with BCG and upregulate cathelicidin to defend against BCG by inhibiting its internalization. Blocking the action of cathelicidin may increase the internalization and effectiveness of BCG in reducing bladder cancer cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

PfSMAD4 plays a role in biomineralization and can transduce bone morphogenetic protein-2 signals in the pearl oyster Pinctada fucata.

PubMed

Zhao, Mi; Shi, Yu; He, Maoxian; Huang, Xiande; Wang, Qi

2016-04-26

Mollusca is the second largest phylum in nature. The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. SMAD4 is an intracellular transmitter in the BMP signaling pathway in mammals, and some genomic data show SMAD4's involvement in BMP signaling in invertebrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested. In this study, we identified a SMAD4 gene (hereafter designated PfSMAD4) in pearl oyster Pinctada fucata. Bioinformatics analysis of PfSMAD4 showed high identity with its orthologs. PfSMAD4 was located in the cytoplasm in immunofluorescence assays and analyses of PfSMAD4 mRNA in tissues and developmental stages showed high expression in ovaries and D-shaped larvae. An RNA interference experiment, performed by PfSMAD4 double-stranded RNA (dsRNA) injection, demonstrated inhibition not only of nacre growth but also organic sheet formation with a decrease in PfSMAD4 expression. A knockdown experiment using PfBMP2 dsRNA showed decreased PfBMP2 and PfSMAD4 mRNA and irregular crystallization of the nacreous layer using scanning electron microscopy. In co-transfection experiments, PfBMP2-transactivated reporter constructs contained PfSMAD4 promoter sequences. Our results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provides important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tashiro, Kanae; Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka; Shishido, Mayumi

2014-01-03

Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Westernmore » blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.« less

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

PubMed Central

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-01-01

ABSTRACT Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes. PMID:27433972

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

PubMed

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-10-01

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

PRDM1 expression via human parvovirus B19 infection plays a role in the pathogenesis of Hashimoto thyroiditis.

PubMed

Wang, Lu; Zhang, Wei-Ping; Yao, Li; Zhang, Wei; Zhu, Jin; Zhang, Wei-Chen; Zhang, Yue-Hua; Wang, Zhe; Yan, Qing-Guo; Guo, Ying; Fan, Lin-Ni; Liu, Yi-Xiong; Huang, Gao-Sheng

2015-12-01

Ectopic lymphoid follicle infiltration is a key event in Hashimoto thyroiditis (HT). Positive regulatory domain zinc finger protein 1 (PRDM1), which is induced by antigen stimulation, can regulate all lymphocyte lineages. Several groups independently demonstrated that human parvovirus B19 (PVB19) is closely associated with HT. Hence, we determined whether PRDM1 is expressed in HT thyroid tissue and whether there is any correlation between PRDM1 expression and PVB19 in the pathogenesis of HT. We detected PRDM1 expression in HT (n = 86), normal thyroid tissue (n = 30), and nontoxic nodular goiter (n = 20) samples using immunohistochemistry. We also detected PVB19 protein in HT samples in a double-blind manner and analyzed the correlation between the 2 proteins using immunofluorescence confocal detection and coimmunoprecipitation. Furthermore, we detected changes of the expression levels of PRDM1 and PVB19 in transfected primary thyroid follicular epithelial cells using real-time quantitative polymerase chain reaction. We found that PRDM1 protein is significantly highly expressed in the injured follicular epithelial cells in HT (83/86 cases) than in normal thyroid cells (0/30 cases) or in nontoxic nodular goiter cells (0/20 cases) (P < .001). In HT, the PRDM1 expression pattern was the same as that of PVB19, whereas PRDM1 and PVB19 were coexistent in the involved epithelial cells. Statistical analysis showed a significant correlation between PRDM1 and PVB19 (P < .001). In addition, primary thyroid epithelial cells also showed PRDM1 up-regulation after PVB19 NS1 transfection. Our findings suggest a previously unrecognized role of PRDM1 and PVB19 in the pathogenesis of HT. Copyright © 2015 Elsevier Inc. All rights reserved.

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.

PubMed

Schläfli, A M; Berezowska, S; Adams, O; Langer, R; Tschan, M P

2015-05-05

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

The polyphenol oleuropein aglycone protects TgCRND8 mice against Aß plaque pathology.

PubMed

Grossi, Cristina; Rigacci, Stefania; Ambrosini, Stefano; Ed Dami, Teresa; Luccarini, Ilaria; Traini, Chiara; Failli, Paola; Berti, Andrea; Casamenti, Fiorella; Stefani, Massimo

2013-01-01

The claimed beneficial effects of the Mediterranean diet include prevention of several age-related dysfunctions including neurodegenerative diseases and Alzheimer-like pathology. These effects have been related to the protection against cognitive decline associated with aging and disease by a number of polyphenols found in red wine and extra virgin olive oil. The double transgenic TgCRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 1.5 and 4, and age-matched wild type control mice were used to examine in vivo the effects of 8 weeks dietary supplementation of oleuropein aglycone (50 mg/kg of diet), the main polyphenol found in extra virgin olive oil. We report here that dietary supplementation of oleuropein aglycone strongly improves the cognitive performance of young/middle-aged TgCRND8 mice, a model of amyloid-ß deposition, respect to age-matched littermates with un-supplemented diet. Immunofluorescence analysis of cerebral tissue in oleuropein aglycone-fed transgenic mice showed remarkably reduced ß-amyloid levels and plaque deposits, which appeared less compact and "fluffy"; moreover, microglia migration to the plaques for phagocytosis and a remarkable reduction of the astrocyte reaction were evident. Finally, oleuropein aglycone-fed mice brain displayed an astonishingly intense autophagic reaction, as shown by the increase of autophagic markers expression and of lysosomal activity. Data obtained with cultured cells confirmed the latter evidence, suggesting mTOR regulation by oleuropein aglycone. Our results support, and provide mechanistic insights into, the beneficial effects against Alzheimer-associated neurodegeneration of a polyphenol enriched in the extra virgin olive oil, a major component of the Mediterranean diet.

[In situ visual imaging of oral squamous cell carcinoma in mice by using near-infrared quantum dots conjugated with arginine-glycine-aspartic acid peptide fluorescent probes].

PubMed

Yunlong, Bai; Hao, Huang; Kai, Yang; Hong, Tang

2014-10-01

To investigate in situ visualization using near-infrared quantum dots (QDs) conjugated with arginine- glycine-aspartic acid (ROD) peptide fluorescent probes in oral squamous cell carcinoma (08CC). QDs with emission wavelength of 800 nm (QD800) were conjugated with RGD peptides to produce QD800-RGD fluorescent probes. Human OSCC cell line BcaCD885 was inoculated in nude mice cheeks to establish OSCC mouse models. Frozen BcaCD885 tumor slices were immunofluorescence double stained by using QD800-RGD and CD105 monoclonal antibody and were observed using a laser scanning confocal microscope. QD800-RGD was injected into the OSCC models through the tail veins, and the in situ visualization was analyzed at different time points. The mice were sacrificed 12 h after injection to isolate tumors for the ex vivo analysis of probe localization in the tumors. QD800-RGD specifically targeted the integrin avβ3 expressed in the endothelial cells of tumor angiogenic vessels in vitro and in vivo, producing clear tumor fluorescence images after intravenous injection. The most complete tumor images with maximal signal-to-noise ratios were observed 0.5 h to 6 h after injection of the probe and significantly reduced 9 h after the injection. However, the tumor image was still clearly visible at 12 h. Using intravenously injected QD800-RGD generates high quality OSCC images when integrin avβ3, which is expressed in the endothelial cells of tumor angiogenic vessels, is used as the target. The technique offers great potential in the diagnosis and individual treatment of OSCC.

Glucose transporters are expressed in taste receptor cells

PubMed Central

Merigo, Flavia; Benati, Donatella; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

2011-01-01

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism. PMID:21592100

Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp 'Golden glue'.

PubMed

Mondal, Nandan Kumar; Ghosh, Sreenita; Ray, Manas Ranjan

2011-04-03

Genotoxicity of glue sniffing/huffing and tobacco use has been examined in 302 street boys (median age 13 years) and 50 age-matched control school boys who were neither tobacco nor glue users. All the street boys were tobacco users. In addition, 155 were addicted to gasp an industrial adhesive popularly known as 'Golden glue'. Micronucleus (MN) frequency was determined as a measure of chromosomal breakage in exfoliated buccal epithelial cells (BECs) and DNA double strand breaks were quantitatively assessed by counting γ-H2AX foci using immunofluorescence microscopy. Micronucleated cell frequencies (MCFs) in BEC of glue non-addicted (only tobacco) and addicted (tobacco plus glue) street boys were 1.87 ± 1.06‰ and 4.04 ± 2.55‰ respectively, which were significantly higher than that of control (0.32 ± 0.11‰, p<0.0001). Similarly, the numbers γ-H2AX foci in nuclei of BEC were 2.3- and 5.2-times more than control in glue non-addicted and addicted street boys respectively (p<0.0001). Spearman's rank correlation revealed a strong positive association between years of glue addiction with MCFs and γ-H2AX foci numbers, and the association between glue addiction and chromosomal and DNA damage remained positive and significant after controlling income, spending on addiction and loss of appetite as potential confounders in multivariate logistic regression analysis. Thus, addiction to tobacco among the street children in India is associated with chromosomal and DNA damage in BECs and the severity of these changes is significantly increased by the habit of sniffing/huffing of industrial glue. Copyright © 2011 Elsevier B.V. All rights reserved.

P63 EXPRESSION LEVELS IN SIDE POPULATION AND LOW LIGHT SCATTERING OCULAR SURFACE EPITHELIAL CELLS

PubMed Central

Epstein, Seth P; Wolosin, J. Mario; Asbell, Penny A

2005-01-01

Purpose Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell–free regions, we examined p63 expression in these stem cell–associated cohorts compared with their controls. Methods Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. Results All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. Conclusions Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health. PMID:17057802

Upregulation of autophagy and glycolysis markers in keloid hypoxic-zone fibroblasts: Morphological characteristics and implications.

PubMed

Ryoko, Okuno; Ito, Yuko; Eid, Nabil; Otsuki, Yoshinori; Kondo, Yoichi; Ueda, Koichi

2018-05-29

Keloid is a fibro-proliferative skin disorder with tumor-like behavior and dependence on anaerobic glycolysis (the Warburg effect), but its exact pathogenesis is unknown. Although autophagy is widely accepted as a lysosomal pathway for cell survival and cellular homeostasis (specifically upon exposure to stressors such as hypoxia), very few studies have investigated the involvement of autophagy and related glycolytic effectors in keloidogenesis. Here the authors examined the expression and cellular localization of autophagy proteins (LC3, pan-cathepsin), glycolytic markers (LDH, MCT1, MCT4) and the transcription factor HIF isoforms in human keloid samples using immunohistochemical analysis and double-labeling immunofluorescence methods. Based on H&E staining and expression of CD31, keloids were compartmentalized into hypoxic central and normoxic marginal zones. Vimentin-expressing fibroblasts in the central zone exhibited greater autophagy than their marginal-zone counterparts, as evidenced by increased LC3 puncta formation and co-localization with lysosomal pan-cathepsin. LDH (a lactate stimulator), MCT4 (a lactate exporter) and HIF-1 α expression levels were also higher in central-zone fibroblasts. Conversely, HIF-2 α expression was upregulated in fibroblasts and endothelial cells of the peripheral zone, while MCT1 was expressed in both zones. Taken together, these observations suggest that upregulation of autophagy and glycolysis markers in keloid hypoxic-zone fibroblasts may indicate a prosurvival mechanism allowing the extrusion of lactate to marginal-zone fibroblasts via metabolic coupling. The authors believe this is the first report on differential expression of autophagic and glycolytic markers in keloid-zone fibroblasts. The study results indicate that autophagy inhibitors and MCT4 blockers may have therapeutic implications in keloid treatment.

5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

PubMed Central

Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C.; Testillano, Pilar S.

2015-01-01

Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs. PMID:26161085

Detection of DNA damage by space radiation in human fibroblast cells flown on the International Space Station

NASA Astrophysics Data System (ADS)

Wu, Honglu; Feiveson, Alan; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao; Wong, Michael

2016-07-01

Although charged particles in space have been detected with radiation detectors on board the spacecraft since the early discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells were kept at 370C in space and fixed on Days 3 and 14 after reaching orbit. After returning to the ground, the fixed cells were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used biomarker for DNA double strand breaks. The 3-dimensional γg-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground controls. To confirm that the foci data from the flight study was actually induced from space radiation exposure, human fibroblast cells were exposed to low- and high-LET protons and high-LET Fe ions on the ground. High-LET protons and Fe ions were found to induce foci of the pattern that were observed in the flown cells.

Association of metallothionein-III with oligodendroglial cytoplasmic inclusions in multiple system atrophy.

PubMed

Pountney, D L; Dickson, T C; Power, J H T; Vickers, J C; West, A J; Gai, W P

2011-01-01

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterised by Parkinsonian and autonomic symptoms and by widespread intracytoplasmic inclusion bodies in oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are comprised of 9-10 nm filaments rich in the protein alpha-synuclein, also found in neuronal inclusion bodies associated with Parkinson's disease. Metallothioneins (MTs) are a class of low-molecular weight (6-7 kDa), cysteine-rich metal-binding proteins the expression of which is induced by heavy metals, glucocorticoids, cytokines and oxidative stress. Recent studies have shown a role for the ubiquitously expressed MT-I/II isoforms in the brain following a variety of stresses, whereas, the function of the brain-specific MT isoform, MT-III, is less clear. MT-III and MT-I/II immunostaining of post-mortem tissue in MSA and normal control human brains showed that the number of MT-III-positive cells is significantly increased in MSA in visual cortex, whereas MT-I/II isoforms showed no significant difference in the distribution of immunopositive cells in MSA compared to normal tissue. GCIs were immunopositive for MT-III, but were immunonegative for the MT-I/II isoforms. Immunofluorescence double labelling showed the co-localisation of alpha-synuclein and MT-III in GCIs in MSA tissue. In isolated GCIs, transmission electron microscopy demonstrated MT-III immunogold labelling of the amorphous material surrounding alpha-synuclein filaments in GCIs. High-molecular weight MT-III species in addition to MT-III monomer were detected in GCIs by Western analysis of the detergent-solubilised proteins of purified GCIs. These results show that MT-III, but not MT-I/II, is a specific component of GCIs, present in abnormal aggregated forms external to the alpha-synuclein filaments.

Pregnancy complicated with Alport syndrome: a good obstetric outcome and failure to diagnose an infant born to a mother with Alport syndrome by umbilical cord immunofluorescence staining.

PubMed

Matsubara, Shigeki; Ueda, Yoshihiko; Takahashi, Hisako; Nagai, Takashi; Kuwata, Tomoyuki; Muto, Shigeaki; Yamaguchi, Takehiko; Takizawa, Toshihiro; Suzuki, Mitsuaki

2009-12-01

Alport syndrome is a familial progressive nephritis. The most frequent type is X-linked Alport syndrome, caused by genetic abnormalities in the alpha 5 chain of type IV collagen. Skin biopsy is a useful tool for diagnosing this disease. It is not well known how this syndrome affects pregnancy and how it is affected by pregnancy, or whether the umbilical cord may provide material for detecting this collagen abnormality. We report a primigravida with Alport syndrome with mild proteinuria who gave birth abdominally to a term male infant without deteriorating renal function during pregnancy. The umbilical cord from not only this infant but also from an Alport (-) control infant showed negative immunofluorescence staining for the alpha 5 chain of type IV collagen. Women with Alport syndrome without renal dysfunction may follow an uneventful obstetrical course until term. The cord may not be suitable for diagnosing Alport syndrome with immunofluorescence staining.

Localization of connexin43 in rat kidney.

PubMed

Barajas, L; Liu, L; Tucker, M

1994-09-01

The localization of connexin43 (Cx 43) in rat kidney was investigated by the indirect immunofluorescence technique with polyclonal antisera raised against Cx 43. Cx 43 is a gap junction protein expressed in a variety of tissues. The typically punctuated gap junction immunofluorescence (GJI) was observed in the renal arterial and arteriolar system. In the renal artery the GJI was concentrated in the media. In the juxtamedullary nephrons, the GJI is particularly abundant in the vascular bundles. There is abundant GJI in the extraglomerular mesangium while in the afferent arteriole GJI appears decreased. Abundant GJI was observed in the inner medullary collecting ducts and pelvic epithelium. The localization of Cx 43 immunofluorescence observed in this study is only in partial agreement with the results of ultrastructural investigations on the distribution of gap junctions in the kidney. An extensive tight junctional system has been demonstrated in the collecting duct system. However, gap junctions have been reported to be absent. Further studies to resolve this discrepancy are required.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

PubMed

Myint, K S; Linthicum, K J; Tanskul, P; Lerdthusnee, K; Vaughn, D W; Manomuth, C; Mongkolsirichaikul, D; Hansukjariya, P; Hastriter, M W

1998-07-01

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

PubMed Central

Lallier, R.; Olivier, G.; Chartier, P.; Turcotte, C.; Dionet, B.; Paulhus, P.

1981-01-01

Presence of the bacterial kidney disease in salmonid fish in Quebec During the summers of 1979 and 1980, wild and hatchery fish were analysed for the presence of the bacterial kidney disease (BKD) agent in salmonid fish in Quebec by the indirect immunofluorescence technique. The causative agent of BKD was detected in all hatcheries tested. Ten to 25% of the fish were positive. The presence of this agent was independent of age and species. We were unable to detect the BKD in fish from the rivers in the northern part of Quebec (over the 50th parallel). The first detection of BKD in Quebec was made in the spring of 1979 in a hatchery. A high mortality occurred in fish over a year old. The majority of the fish were positive by immunofluorescence and the bacteriun was isolated from some fish. Three weeks before the disease appeared, six ponds received fish from this hatchery. No mortality was observed in those ponds and in the middle of the summer the percentage of carrier fish by immunofluorescence was approximately 20%. PMID:7340922

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks.

PubMed

Poojan, Shiv; Kim, Han-Seong; Yoon, Ji-Woon; Sim, Hye Won; Hong, Kyeong-Man

2018-05-20

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Centripetal myosin redistribution in thrombin-stimulated platelets. Relationship to platelet Factor 4 secretion.

PubMed

Painter, R G; Ginsberg, M H

1984-11-01

We have examined the F-actin and myosin distribution in resting and thrombin-activated platelets by double label immunofluorescence microscopy. In resting, discoid platelets, F-actin and myosin staining was distributed in a diffuse pattern throughout the interior of the cell with slight accentuation at the cell periphery. In contrast, platelet factor 4 antigen (PF4) was more centrally localized in a fine punctate distribution which is consistent with its localization in alpha-granules. Within 5 sec after thrombin stimulation both F-actin and myosin staining were increased at the periphery of the now spherical platelets. Subsequently, a myosin-containing spherical structure decreased in diameter closely surrounding a phase-dense central zone. In contrast, F-actin staining continued to be accentuated at the cell periphery and was prominent in filopodia and blebs. As previously shown, PF4 staining was localized after 30 sec within large intracellular masses that corresponded to closed vacuolar structures at the ultrastructural level. Morphometric analysis of electron micrographs showed that formation of these vacuolar structures kinetically paralleled alpha-granule disappearance and preceded PF4 release. These PF4-containing structures translocated to the cell periphery after 1-3 min, where they appeared to fuse with the plasma membrane. Ultrastructural analysis of thin sections showed that the myosin-rich spherical structure spatially and temporally correlated with a band of microfilaments that closely surrounded the organelle-rich central zone of the cell. Morphometric analysis of these micrographs showed that the absolute volume of this central zone decreased with time after thrombin addition, showing a significant change after 15 sec and reaching a maximum value after 3-5 min. Changes in the volume of this compartment kinetically preceded PF4 release. On the basis of these data, we propose that an actomyosin contractile force is generated which centripetally redistributes the myosinrich structure and organelle zone. Conceivably this inward force may not only accelerate granule-granule fusion to form intracellular secretory vacuoles, but may also provide aid in their extrusion toward the platelet plasma membrane.

Trichohyalin is a potential major autoantigen in human alopecia areata.

PubMed

Leung, Man Ching; Sutton, Chris W; Fenton, David A; Tobin, Desmond J

2010-10-01

Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AA-reactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder.

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells.

PubMed

Benavente, R; Reimer, G; Rose, K M; Hügle-Dörr, B; Scheer, U

1988-01-01

After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

Methods to Monitor DNA Repair Defects and Genomic Instability in the Context of a Disrupted Nuclear Lamina.

PubMed

Gonzalo, Susana; Kreienkamp, Ray

2016-01-01

The organization of the genome within the nuclear space is viewed as an additional level of regulation of genome function, as well as a means to ensure genome integrity. Structural proteins associated with the nuclear envelope, in particular lamins (A- and B-type) and lamin-associated proteins, play an important role in genome organization. Interestingly, there is a whole body of evidence that links disruptions of the nuclear lamina with DNA repair defects and genomic instability. Here, we describe a few standard techniques that have been successfully utilized to identify mechanisms behind DNA repair defects and genomic instability in cells with an altered nuclear lamina. In particular, we describe protocols to monitor changes in the expression of DNA repair factors (Western blot) and their recruitment to sites of DNA damage (immunofluorescence); kinetics of DNA double-strand break repair after ionizing radiation (neutral comet assays); frequency of chromosomal aberrations (FISH, fluorescence in situ hybridization); and alterations in telomere homeostasis (Quantitative-FISH). These techniques have allowed us to shed some light onto molecular mechanisms by which alterations in A-type lamins induce genomic instability, which could contribute to the pathophysiology of aging and aging-related diseases.

Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

PubMed

Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

2017-06-01

Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Localization of PPAR isotypes in the adult mouse and human brain

PubMed Central

Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B.; Mayfield, R. Dayne; Harris, R. Adron

2016-01-01

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain. PMID:27283430

Localization of PPAR isotypes in the adult mouse and human brain.

PubMed

Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B; Mayfield, R Dayne; Harris, R Adron

2016-06-10

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain.

Up-regulated BAFF and BAFF receptor expression in patients with intractable temporal lobe epilepsy and a pilocarpine-induced epilepsy rat model.

PubMed

Ma, Limin; Li, Ruohan; Huang, Hao; Yuan, Jinxian; Ou, Shu; Xu, Tao; Yu, Xinyuan; Liu, Xi; Chen, Yangmei

2017-05-01

Some studies have suggested that BAFF and BAFFR are highly expressed in the central nervous system (CNS) and participate in inflammatory and immune associated diseases. However, whether BAFF and BAFFR are involved in the pathogenesis of epilepsy remains unknown. This study aimed to investigate the expression of BAFF and BAFFR proteins in the brains of patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model of TLE to identify possible roles of the BAFF-BAFFR signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, immunohistochemistry, and double-immunofluorescence were performed in this study. The results showed that BAFF and BAFFR expression levels were markedly up-regulated in intractable TLE patients and TLE rats. Moreover, BAFF and BAFFR proteins mainly highly expressed in the membranes and cytoplasms of the dendritic marker MAP2 in the cortex and hippocampus. Therefore, the significant increased in BAFF and BAFFR protein expression in both TLE patients and rats suggest that BAFF and BAFFR may play important roles in regulating the pathogenesis of epilepsy. Copyright © 2017 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

[The role of RKIP mediated ERK pathway in hippocampus neurons injured by electromagnetic radiation].

PubMed

Zuo, Hong-Yan; Wang, De-Wen; Peng, Rui-Yun; Wang, Shui-Ming; Gao, Ya-Bing; Zhang, Zhi-Yi; Xiao, Feng-Jun

2008-07-01

To study the effects of electromagnetic radiation on RKIP and phosphorylated ERK in primary cultured hippocampus neurons. The inhibitor of MEK U0126 was applied to investigate the role of RKIP mediated ERK pathway in radiation injury. Primary hippocampus neurons were cultured in vitro. X-HPM, S-HPM and EMP were taken as radiation source respectively to establish three cell models exposed to electromagnetic radiation. RKIP and phosphorylated ERK were measured by immunofluorescent labelling and laser scanning confocal microscope. Apoptosis and death fraction of the cells were detected by Annexin V-PI double labelling and flow cytometry. After three kinds of electromagnetic radiation, the expression of RKIP in hippocampus neurons decreased but the expression of phosphorylated ERK increased, and its nuclear translocation occurred. No significant differences were seen between radiation groups. Apoptosis and death fraction of the neurons in U0126 pretreatment groups was significantly lower than that in radiation groups but they were still higher than those in sham-radiation group. The excessive activation of RKIP mediated ERK pathway is one of the important mechanisms for the apoptosis and death of hippocampus neurons induced by electromagnetic radiation. U0126 have some protective effects on radiation injury.

Meiotic Recombination in the Giraffe (G. reticulata).

PubMed

Vozdova, Miluse; Fröhlich, Jan; Kubickova, Svatava; Sebestova, Hana; Rubes, Jiri

2017-01-01

Recently, the reticulated giraffe (G. reticulata) was identified as a distinct species, which emphasized the need for intensive research in this interesting animal. To shed light on the meiotic process as a source of biodiversity, we analysed the frequency and distribution of meiotic recombination in 2 reticulated giraffe males. We used immunofluorescence detection of synaptonemal complex protein (SYCP3), meiotic double strand breaks (DSB, marked as RAD51 foci) in leptonema, and crossovers (COs, as MLH1 foci) in pachynema. The mean number of autosomal MLH1 foci per cell (27), which resulted from a single, distally located MLH1 focus observed on most chromosome arms, is one of the lowest among mammalian species analysed so far. The CO/DSB conversion ratio was 0.32. The pseudoautosomal region was localised in the Xq and Yp termini by FISH and showed an MLH1 focus in 83% of the pachytene cells. Chromatin structures corresponding to the nucleolus organiser regions were observed in the pachytene spermatocytes. The results are discussed in the context of known data on meiosis in Cetartiodactyla, depicting that the variation in CO frequency among species of this taxonomic group is mostly associated with their diploid chromosome number. © 2017 S. Karger AG, Basel.

Immunohistochemical detection of autophagy-related microtubule-associated protein 1 light chain 3 (LC3) in the cerebellums of dogs naturally infected with canine distemper virus.

PubMed

Kabak, Y B; Sozmen, M; Yarim, M; Guvenc, T; Karayigit, M O; Gulbahar, M Y

2015-01-01

We investigated the expression of microtubule-associated protein 1 light chain 3 (LC3) protein in the cerebellums of dogs infected with canine distemper virus (CDV) using immunohistochemistry to detect autophagy. The cerebellums of 20 dogs infected with CDV were used. Specimens showing demyelination of white matter were considered to have an acute infection, whereas specimens showing signs of severe perivascular cuffing and demyelination of white matter were classified as having chronic CDV. Cerebellar sections were immunostained with CDV and LC3 antibodies. The cytoplasm of Purkinje cells, granular layer cells, motor neurons in large cerebellar ganglia and some neurons in white matter were positive for the LC3 antibody in both the control and CDV-infected dogs. In the infected cerebellums, however, white matter was immunostained more intensely, particularly the neurons and gemistocytic astrocytes in the demyelinated areas, compared to controls. Autophagy also was demonstrated in CDV-positive cells using double immunofluorescence staining. Our findings indicate that increased autophagy in the cerebellum of dogs naturally infected with CDV may play a role in transferring the virus from cell to cell.

Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato1

PubMed Central

Aoki, Koh; Yamamoto, Miyuki; Wada, Keishiro

1998-01-01

Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts. PMID:9765529

A PCR-Based Method for RNA Probes and Applications in Neuroscience.

PubMed

Hua, Ruifang; Yu, Shanshan; Liu, Mugen; Li, Haohong

2018-01-01

In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore bemore » added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.« less

TRPV2, a capsaicin receptor homologue, is expressed predominantly in the neurotrophin-3-dependent subpopulation of primary sensory neurons.

PubMed

Tamura, S; Morikawa, Y; Senba, E

2005-01-01

TRPV2, a member of transient receptor potential ion channels, responds to high-threshold noxious heat, but neither to capsaicin nor to proton. Although TRPV2 is expressed in medium- to large-sized dorsal root ganglion (DRG) neurons with myelinated fibers in adult rodents, little is known about the neurotrophin dependence of TRPV2-positive neurons in the developing and adult DRGs of mice. In the present study, using immunohistochemistry, we found that TRPV2 was first expressed in DRG neurons at embryonic day (E) 11.5, when neither TRPV1 nor TRPM8 was detected yet. Double-immunofluorescence staining revealed that tyrosine kinase receptor C (TrkC) was expressed in most of TRPV2-positive DRG neurons at E11.5 and E13.5. In addition, the percentage of TRPV2-positive neurons in the total DRG neurons at E13.5 reached the same as that of adulthood. In adult DRGs, TrkC and Ret were expressed in 68% and 25% of TRPV2-positive neurons, respectively. These results suggest that TRPV2 is expressed predominantly in the NT-3-dependent subpopulation of DRG neurons throughout development and in adult mice.

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

PubMed

Takenaka, Nobuyuki; Nihata, Yuma; Ueda, Sho; Satoh, Takaya

2017-11-01

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1.

PubMed

Kerkar, N; Ma, Y; Hussain, M; Muratori, L; Targett, C; Williams, R; Bianchi, F B; Mieli-Vergani, G; Vergani, D

1999-03-04

Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

PubMed Central

Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

2014-01-01

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation. PMID:25489864

Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

PubMed Central

Premer, Courtney; Lamondin, Courtney; Mitzey, Ann; Speth, Robert C.; Brownfield, Mark S.

2013-01-01

Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors. PMID:23573410

Design and Development of a Miniaturized Double Latching Solenoid Valve for the Sample Analysis at Mars Instrument Suite

NASA Technical Reports Server (NTRS)

Smith, James T.

2008-01-01

The development of the in-house Miniaturized Double Latching Solenoid Valve, or Microvalve, for the Gas Processing System (GPS) of the Sample Analysis at Mars (SAM) instrument suite is described. The Microvalve is a double latching solenoid valve that actuates a pintle shaft axially to hermetically seal an orifice. The key requirements and the design innovations implemented to meet them are described.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

In situ localization of nucleolin in the plant nucleolar matrix.

PubMed

Minguez, A; Moreno Diaz de la Espina, S

1996-01-10

The analysis of isolated nucleolar matrices from onion cells by light and electron microscopy, 2-D separation of proteins, and confocal microscopy has confirmed the existence of an organized nucleolar matrix with a complex protein composition to which are attached the insoluble processing complexes. In the present work, we present evidence from immunoblotting, immunofluorescence, immunogold labeling, and preferential cytochemical staining with bismuth salts that an insoluble fraction of the multifunctional protein nucleolin, is a component of the onion nucleolar matrix, and analyse its ultrastructural distribution in the described domains of the matrix.

Logarithmic amplifiers.

PubMed

Gandler, W; Shapiro, H

1990-01-01

Logarithmic amplifiers (log amps), which produce an output signal proportional to the logarithm of the input signal, are widely used in cytometry for measurements of parameters that vary over a wide dynamic range, e.g., cell surface immunofluorescence. Existing log amp circuits all deviate to some extent from ideal performance with respect to dynamic range and fidelity to the logarithmic curve; accuracy in quantitative analysis using log amps therefore requires that log amps be individually calibrated. However, accuracy and precision may be limited by photon statistics and system noise when very low level input signals are encountered.

Increased centrosome number in BRCA-related breast cancer specimens determined by immunofluorescence analysis.

PubMed

Watanabe, Gou; Chiba, Natsuko; Nomizu, Tadashi; Furuta, Akihiko; Sato, Kaolu; Miyashita, Minoru; Tada, Hiroshi; Suzuki, Akihiko; Ohuchi, Noriaki; Ishida, Takanori

2018-06-01

BRCA-related breast carcinoma can be prevented through prophylactic surgery and an intensive follow-up regimen. However, BRCA genetic tests cannot be routinely performed, and some BRCA mutations could not be defined as deleterious mutations or normal variants. Therefore, an easy functional assay of BRCA will be useful to evaluate BRCA status. As it has been reported that BRCA functions in the regulation of centrosome number, we focused on centrosome number in cancer tissues. Here, 70 breast cancer specimens with known BRCA status were analyzed using immunofluorescence of γ-tubulin (a marker of centrosome) foci. The number of foci per cell was higher in cases with BRCA mutation compared to wild-type cases, that is, 1.9 (95% confidence interval [CI], 1.5-2.3) vs 0.5 (95% CI, 0.2-0.8) (P < .001). Specifically, foci numbers per cell in BRCA1 and BRCA2 mutation cases were 1.2 (95% CI, 0.6-1.8) and 2.2 (95% CI, 1.7-2.6), respectively, both higher than those in wild-type cases (P = .042 and P < .0001, respectively). The predictive value of γ-tubulin foci as determined by area under the curve (AUC = 0.86) for BRCA status was superior to BRCAPRO (AUC = 0.69), Myriad Table (AUC = 0.61), and KOHBRA BRCA risk calculator (AUC = 0.65) pretest values. The use of γ-tubulin foci to predict BRCA status had sensitivity = 83% (19/23), specificity = 89% (42/47), and positive predictive value = 77% (20/26). Thus, γ-tubulin immunofluorescence, a functional assessment of BRCA, can be used as a new prospective test of BRCA status. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

Fibrillin-1 Expression Is Decreased in the Diaphragmatic Muscle Connective Tissue of Nitrofen-Induced Congenital Diaphragmatic Hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2017-02-01

Introduction  Diaphragmatic morphogenesis depends on proper formation of muscle connective tissue (MCT) and underlying extracellular matrix (ECM). Fibrillin-1 is an essential ECM protein and crucial for the structural integrity of MCT in the developing diaphragm. Recently, mutations in the fibrillin-1 gene (FBN1) have been identified in cases of congenital diaphragmatic hernia (CDH), thus suggesting that alterations in FBN1 gene expression may lead to diaphragmatic defects. We designed this study to investigate the hypothesis that the diaphragmatic expression of fibrillin-1 is decreased in the MCT of nitrofen-induced CDH. Materials and Methods  Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms ( n  = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Laser-capture microdissection was used to obtain diaphragmatic tissue cells. Gene expression levels of FBN1 were analyzed by qRT-PCR. Immunofluorescence-double-staining for fibrillin-1 and the mesenchymal marker Gata4 was performed to evaluate protein expression and localization. Results  Relative mRNA expression of FBN1 was significantly decreased in pleuroperitoneal folds on D13 (3.39 ± 1.29 vs. 5.47 ± 1.92; p  < 0.05), developing diaphragms on D15 (2.48 ± 0.89 vs. 4.03 ± 1.62; p  < 0.05), and fully muscularized diaphragms on D18 (2.49 ± 0.69 vs. 3.93 ± 1.55; p  < 0.05) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning microscopy revealed markedly diminished fibrillin-1 immunofluorescence mainly in MCT, associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. Conclusions  Decreased expression of fibrillin-1 during morphogenesis of the fetal diaphragm may disrupt mesenchymal cell proliferation, causing malformed MCT and thus resulting in diaphragmatic defects in the nitrofen-induced CDH model. Georg Thieme Verlag KG Stuttgart · New York.

[Effect of lead-exposed astrocytes on neuronal synaptic formation].

PubMed

Cui, Yan; Li, Tingting; Yu, Haiyang; Liao, Yingjun; Jin, Yaping

2014-09-01

To investigate the effect of lead-exposed astrocyte conditioned medium (ACM) on the synaptic formation of neurons and to provide reference for the mechanism of lead neurotoxicity. Astrocytes were cultured in the medium containing 50, 100, 200, 400, and 800 µmol/L lead acetate for 72 h. Alamar Blue was used to assess the cell viability of astrocytes, and then ACM was collected. Primarily cultured neurons were divided into six groups: pure culture group, non-glutamic acid (Glu)-induced ACM treatment group, Glu-induced lead-free ACM treatment group, and Glu-induced 50, 100, and 200 µmol/L lead acetate-exposed ACM treatment groups. Neurons were collected after being cultured in ACM for 24, 48, or 72 h. The content of synaptophysin (SYP) in neurons was determined by Western blot. The SYP expression in neurons was measured by immunofluorescence after being cultured in ACMfor 72 h. In all lead-exposed groups, the cell viability of astrocytes declined with increasing concentration of lead (P < 0.05). The Western blot showed that compared with the pure culture group, the non-Glu-induced ACM treatment group and Glu-induced lead- free ACM treatment group had significantly increased content of SYP in neurons (P < 0.01); compared with the non-Glu-induced ACM treatment group, the Glu-induced ACM treatment groups had significantly reduced SYP expression in neurons (P < 0.05); compared with the Glu-induced lead-free ACM treatment group, all lead-exposed ACM treatment groups had the content of SYP in neurons significantly reduced with increasing concentration of lead after 72-h culture (P < 0.01), the 200 µmol/L lead-exposed ACM treatment group had significantly reduced content of SYP in neurons after 48-h culture (P < 0.01), and all lead-exposed ACM treatment groups showed no significant changes in the content of SYP in neurons after 24-h culture. Double-labeling immunofluorescence of SYP showed that all lead-exposed ACM treatment groups had a significant decrease in the number of SYP-fluorescent particles after 72-h culture (P < 0.05). Astrocytes promote synaptic formation of neurons, which may be inhibited during lead exposure.

Breast-implant texturing associated with delamination of capsular layers: A histological analysis of the double capsule phenomenon.

PubMed

Efanov, J I; Giot, J P; Fernandez, J; Danino, M A

2017-06-01

Macro-texturing of breast implants was developed with the double goal of improving implant stabilization within the breast cavity and decreasing the rate of capsular contractures. However, recent evidence suggests that double capsular formation, a potentially worrisome phenomenon associated with late seromas and biofilms, occurs with preponderance in macro-textured implants. Our objective was to analyze histologically different regions of double capsules to determine if they are more prone to mechanical movements. A prospective analysis including patients undergoing second-stage expander to definitive breast-implant reconstruction post-mastectomy was conducted after intraoperative identification of the double capsule phenomenon. Two samples were collected from each capsules around the implant, located centrally and laterally. The specimens were sent for histological analysis by the institution's pathologist. In total, 10 patients were identified intraoperatively with partial double capsule phenomenon. Among samples retrieved from the lateral aspect of the breast implant, all were associated with delamination and fractures in the collagen matrix of the double capsules. This phenomenon was not observed in any sample from the dome of the breast. Breast-implant macro-texturing plays an important role on delamination of capsules on lateral portions of the breast, which may have an etiologic role in double capsule formation. Manufacturing implants with macro-texturing on one side and smooth surface on the other could diminish mechanical shear forces responsible for these findings. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Numerical analysis of the unintegrated double gluon distribution

NASA Astrophysics Data System (ADS)

Elias, Edgar; Golec-Biernat, Krzysztof; Staśto, Anna M.

2018-01-01

We present detailed numerical analysis of the unintegrated double gluon distribution which includes the dependence on the transverse momenta of partons. The unintegrated double gluon distribution was obtained following the Kimber-Martin-Ryskin method as a convolution of the perturbative gluon splitting function with the collinear integrated double gluon distribution and the Sudakov form factors. We analyze the dependence on the transverse momenta, longitudinal momentum fractions and hard scales. We find that the unintegrated gluon distribution factorizes into a product of two single unintegrated gluon distributions in the region of small values of x, provided the splitting contribution is included and the momentum sum rule is satisfied.

The double universal joint wrist on a manipulator: Solution of inverse position kinematics and singularity analysis

NASA Technical Reports Server (NTRS)

Williams, Robert L., III

1992-01-01

This paper presents three methods to solve the inverse position kinematics position problem of the double universal joint attached to a manipulator: (1) an analytical solution for two specific cases; (2) an approximate closed form solution based on ignoring the wrist offset; and (3) an iterative method which repeats closed form position and orientation calculations until the solution is achieved. Several manipulators are used to demonstrate the solution methods: cartesian, cylindrical, spherical, and an anthropomorphic articulated arm, based on the Flight Telerobotic Servicer (FTS) arm. A singularity analysis is presented for the double universal joint wrist attached to the above manipulator arms. While the double universal joint wrist standing alone is singularity-free in orientation, the singularity analysis indicates the presence of coupled position/orientation singularities of the spherical and articulated manipulators with the wrist. The cartesian and cylindrical manipulators with the double universal joint wrist were found to be singularity-free. The methods of this paper can be implemented in a real-time controller for manipulators with the double universal joint wrist. Such mechanically dextrous systems could be used in telerobotic and industrial applications, but further work is required to avoid the singularities.

Characterization of the Bm61 of the Bombyx mori nucleopolyhedrovirus.

PubMed

Shen, Hongxing; Chen, Keping; Yao, Qin; Zhou, Yang

2009-07-01

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.

Comparison of growth inhibition and immunofluorescence tests in serotyping clinical isolates of Ureaplasma urealyticum.

PubMed Central

Piot, P

1977-01-01

Typestrains of the eight serotypes of Ureaplasma urealyticum and 39 genital isolates were typed by the growth inhibition (GI) and indirect immunofluorescence (IF) tests. The GI test proved to be very specific and simple, demanding large volumes of serum. It was less sensitive than the IF test, which produced more cross-reactions, was economical in serum, and able to detect mixed infections. Of the 39 isolates, 27 were serotyped by GI and 34 by IF. Mixed cultures occurred in as many as 36% of the isolates. PMID:326348

Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

NASA Astrophysics Data System (ADS)

Decho, Alan W.; Beckman, Erin M.; Chandler, G. Thomas; Kawaguchi, Tomohiro

2008-06-01

An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.

ISOLATION AND DETECTION OF GIARDIA CYSTS FROM WATER USING DIRECT IMMUNOFLUORESCENCE.

USGS Publications Warehouse

Sorenson, Stephen K.; Riggs, John L.; Dileanis, Peter D.; Suk, Thomas J.

1986-01-01

A water-sampling apparatus used for the isolation and detection of Giardia cysts in water has been designed and tested. The sampling apparatus uses one of a variety of pumps or waterline pressure to move water through a filter. Two of the optional pumps are lightweight enough to make the apparatus portable and thus suitable for sampling in remote areas. This technique of sample processing produces good cyst recovery in much less time than is required with previously established methods. Giardia cysts are identified using direct immunofluorescence.

Epidermis area detection for immunofluorescence microscopy

NASA Astrophysics Data System (ADS)

Dovganich, Andrey; Krylov, Andrey; Nasonov, Andrey; Makhneva, Natalia

2018-04-01

We propose a novel image segmentation method for immunofluorescence microscopy images of skin tissue for the diagnosis of various skin diseases. The segmentation is based on machine learning algorithms. The feature vector is filled by three groups of features: statistical features, Laws' texture energy measures and local binary patterns. The images are preprocessed for better learning. Different machine learning algorithms have been used and the best results have been obtained with random forest algorithm. We use the proposed method to detect the epidermis region as a part of pemphigus diagnosis system.

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

PubMed

Popp, Vanessa; Gerlach, Katharina; Mott, Stefanie; Turowska, Agnieszka; Garn, Holger; Atreya, Raja; Lehr, Hans-Anton; Ho, I-Cheng; Renz, Harald; Weigmann, Benno; Neurath, Markus F

2017-01-01

GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

3D multiplexed immunoplasmonics microscopy

NASA Astrophysics Data System (ADS)

Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

2016-07-01

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence. Electronic supplementary information (ESI) available: Characterization of functionalized nanoparticles by UV-visible-NIR spectroscopy, standard dark field microscopy and reflected light microscopy. Immunofluorescence of cells. See DOI: 10.1039/c6nr01257d

Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells.

PubMed

Voigt, Jörn; Krause, Christopher; Rohwäder, Edda; Saschenbrecker, Sandra; Hahn, Melanie; Danckwardt, Maick; Feirer, Christian; Ens, Konstantin; Fechner, Kai; Barth, Erhardt; Martinetz, Thomas; Stöcker, Winfried

2012-01-01

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Association of p60c-src with endosomal membranes in mammalian fibroblasts

PubMed Central

1992-01-01

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three- dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI- MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking. PMID:1378446

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells

PubMed Central

Voigt, Jörn; Krause, Christopher; Rohwäder, Edda; Saschenbrecker, Sandra; Hahn, Melanie; Danckwardt, Maick; Feirer, Christian; Ens, Konstantin; Fechner, Kai; Barth, Erhardt; Martinetz, Thomas; Stöcker, Winfried

2012-01-01

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability. PMID:23251220

Pathological Gleason prediction through gland ring morphometry in immunofluorescent prostate cancer images

NASA Astrophysics Data System (ADS)

Scott, Richard; Khan, Faisal M.; Zeineh, Jack; Donovan, Michael; Fernandez, Gerardo

2016-03-01

The Gleason score is the most common architectural and morphological assessment of prostate cancer severity and prognosis. There have been numerous quantitative techniques developed to approximate and duplicate the Gleason scoring system. Most of these approaches have been developed in standard H and E brightfield microscopy. Immunofluorescence (IF) image analysis of tissue pathology has recently been proven to be extremely valuable and robust in developing prognostic assessments of disease, particularly in prostate cancer. There have been significant advances in the literature in quantitative biomarker expression as well as characterization of glandular architectures in discrete gland rings. In this work we leverage a new method of segmenting gland rings in IF images for predicting the pathological Gleason; both the clinical and the image specific grade, which may not necessarily be the same. We combine these measures with nuclear specific characteristics as assessed by the MST algorithm. Our individual features correlate well univariately with the Gleason grades, and in a multivariate setting have an accuracy of 85% in predicting the Gleason grade. Additionally, these features correlate strongly with clinical progression outcomes (CI of 0.89), significantly outperforming the clinical Gleason grades (CI of 0.78). This work presents the first assessment of morphological gland unit features from IF images for predicting the Gleason grade.

Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China.

PubMed

Cheng, Shunfeng; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

2006-08-01

Lymphocystis disease virus (LCDV) can infect, both naturally and experimentally, about 100 different teleost fish species. In this study, LCDV was purified using differential and gradient centrifugation from skin tumours of Japanese flounder Paralichthys olivaceus. A panel of five monoclonal antibodies (Mabs) against LCDV were produced by immunization of Balb/c mice with purified virus preparations. Analysed by the indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), Western blot and immunogold electron microscopy (IEM), they showed specificity for LCDV. Immunofluorescent studies showed that the specific fluorescence signals appeared at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped. Western blot analysis demonstrated that two Mabs 1D7 and 2B6 reacted specifically to a single protein with an approximately molecular weight of 116kDa, Mab 3G3 reacted with two LCDV proteins at molecular mass of approximately 116 and 90kDa. Immunogold transmission electron-microscopy provided visualized evidence that the epitopes recognized by these Mabs were located on the outer surface of virions. The Mabs characterized should prove useful for developing LCDV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Two-step rating-based 'double-faced applicability' test for sensory analysis of spread products as an alternative to descriptive analysis with trained panel.

PubMed

Kim, In-Ah; den-Hollander, Elyn; Lee, Hye-Seong

2018-03-01

Descriptive analysis with a trained sensory panel has thus far been the most well defined methodology to characterize various products. However, in practical terms, intensive training in descriptive analysis has been recognized as a serious defect. To overcome this limitation, various novel rapid sensory profiling methodologies have been suggested in the literature. Among these, attribute-based methodologies such as check-all-that-apply (CATA) questions showed results comparable to those of conventional sensory descriptive analysis. Kim, Hopkinson, van Hout, and Lee (2017a, 2017b) have proposed a novel attribute-based methodology termed the two-step rating-based 'double-faced applicability' test with a novel output measure of applicability magnitude (d' A ) for measuring consumers' product usage experience throughout various product usage stages. In this paper, the potential of the two-step rating-based 'double-faced applicability' test with d' A was investigated as an alternative to conventional sensory descriptive analysis in terms of sensory characterization and product discrimination. Twelve commercial spread products were evaluated using both conventional sensory descriptive analysis with a trained sensory panel and two-step rating-based 'double-faced applicability' test with an untrained sensory panel. The results demonstrated that the 'double-faced applicability' test can be used to provide a direct measure of the applicability magnitude of sensory attributes of the samples tested in terms of d' A for sensory characterization of individual samples and multiple sample comparisons. This suggests that when the appropriate list of attributes to be used in the questionnaire is already available, the two-step rating-based 'double-faced applicability' test with d' A can be used as a more efficient alternative to conventional descriptive analysis, without requiring any intensive training process. Copyright © 2017 Elsevier Ltd. All rights reserved.

Did Shakespeare write double falsehood? Identifying individuals by creating psychological signatures with text analysis.

PubMed

Boyd, Ryan L; Pennebaker, James W

2015-05-01

More than 100 years after Shakespeare's death, Lewis Theobald published Double Falsehood, a play supposedly sourced from a lost play by Shakespeare and John Fletcher. Since its release, scholars have attempted to determine its true authorship. Using new approaches to language and psychological analysis, we examined Double Falsehood and the works of Theobald, Shakespeare, and Fletcher. Specifically, we created a psychological signature from each author's language and statistically compared the features of each signature with those of Double Falsehood's signature. Multiple analytic approaches converged in suggesting that Double Falsehood's psychological style and content architecture predominantly resemble those of Shakespeare, showing some similarity with Fletcher's signature and only traces of Theobald's. Closer inspection revealed that Shakespeare's influence is most apparent early in the play, whereas Fletcher's is most apparent in later acts. Double Falsehood has a psychological signature consistent with that expected to be present in the long-lost play The History of Cardenio, cowritten by Shakespeare and Fletcher. © The Author(s) 2015.

Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

PubMed

Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

2016-02-01

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Involvement of pro-inflammatory cytokines and growth factors in the pathogenesis of Dupuytren's contracture: a novel target for a possible future therapeutic strategy?

PubMed

Bianchi, Enrica; Taurone, Samanta; Bardella, Lia; Signore, Alberto; Pompili, Elena; Sessa, Vincenzo; Chiappetta, Caterina; Fumagalli, Lorenzo; Di Gioia, Cira; Pastore, Francesco S; Scarpa, Susanna; Artico, Marco

2015-10-01

Dupuytren's contracture (DC) is a benign fibro-proliferative disease of the hand causing fibrotic nodules and fascial cords which determine debilitating contracture and deformities of fingers and hands. The present study was designed to characterize pro-inflammatory cytokines and growth factors involved in the pathogenesis, progression and recurrence of this disease, in order to find novel targets for alternative therapies and strategies in controlling DC. The expression of pro-inflammatory cytokines and of growth factors was detected by immunohistochemistry in fibrotic nodules and normal palmar fascia resected respectively from patients affected by DC and carpal tunnel syndrome (CTS; as negative controls). Reverse transcription (RT)-PCR analysis and immunofluorescence were performed to quantify the expression of transforming growth factor (TGF)-β1, interleukin (IL)-1β and vascular endothelial growth factor (VEGF) by primary cultures of myofibroblasts and fibroblasts isolated from Dupuytren's nodules. Histological analysis showed high cellularity and high proliferation rate in Dupuytren's tissue, together with the presence of myofibroblastic isotypes; immunohistochemical staining for macrophages was completely negative. In addition, a strong expression of TGF-β1, IL-1β and VEGF was evident in the extracellular matrix and in the cytoplasm of fibroblasts and myofibroblasts in Dupuytren's nodular tissues, as compared with control tissues. These results were confirmed by RT-PCR and by immunofluorescence in pathological and normal primary cell cultures. These preliminary observations suggest that TGF-β1, IL-1β and VEGF may be considered potential therapeutic targets in the treatment of Dupuytren's disease (DD). © 2015 Authors; published by Portland Press Limited.

Size and deformability based separation of circulating tumor cells from castrate resistant prostate cancer patients using resettable cell traps.

PubMed

Qin, Xi; Park, Sunyoung; Duffy, Simon P; Matthews, Kerryn; Ang, Richard R; Todenhöfer, Tilman; Abdi, Hamid; Azad, Arun; Bazov, Jenny; Chi, Kim N; Black, Peter C; Ma, Hongshen

2015-05-21

The enumeration and capture of circulating tumor cells (CTCs) are potentially of great clinical value as they offer a non-invasive means to access tumor materials to diagnose disease and monitor treatment efficacy. Conventional immunoenrichment of CTCs may fail to capture cells with low surface antigen expression. Micropore filtration presents a compelling label-free alternative that enriches CTCs using their biophysical rather than biochemical characteristics. However, this strategy is prone to clogging of the filter microstructure, which dramatically reduces the selectivity after processing large numbers of cells. Here, we use the resettable cell trap (RCT) mechanism to separate cells based on their size and deformability using an adjustable aperture that can be periodically cleared to prevent clogging. After separation, the output sample is stained and analyzed using multi-spectral analysis, which provides a more sensitive and unambiguous method to identify CTC biomarkers than traditional immunofluorescence. We tested the RCT device using blood samples obtained from 22 patients with metastatic castrate-resistant prostate cancer while comparing the results with the established CellSearch® system. The RCT mechanism was able to capture ≥5 CTCs in 18/22 (82%) patients with a mean count of 257 in 7.5 ml of whole blood, while the CellSearch system found ≥5 CTCs in 9/22 (41%) patients with a mean count of 25. The ~10× improvement in the CTC capture rate provides significantly more materials for subsequent analysis of these cells such as immunofluorescence, propagation by tissue culture, and genetic profiling.

Increasing the accuracy and scalability of the Immunofluorescence Assay for Epstein Barr Virus by inferring continuous titers from a single sample dilution.

PubMed

Goh, Sherry Meow Peng; Swaminathan, Muthukaruppan; Lai, Julian U-Ming; Anwar, Azlinda; Chan, Soh Ha; Cheong, Ian

2017-01-01

High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution. In the first part of our study, 2 blinded evaluators used a set of reference titer standards to perform independent re-evaluations of historical samples with known titers. Besides exhibiting high inter-evaluator agreement, both evaluators were also in high concordance with historical titers, thus validating the accuracy of the reference titer standards. In the second part of the study, the reference titer standards were IFA-processed and assigned an 'EBV Score' using image analysis. A log-linear relationship between titers and EBV Score was observed. This relationship was preserved even when images were acquired and analyzed 3days post-IFA. We conclude that image analysis of IFA-processed samples can be used to infer a continuous EBV titer with just a single dilution of NPC-positive patient sera. This work opens new possibilities for improving the accuracy and scalability of IFA in the context of clinical screening. Copyright © 2016. Published by Elsevier B.V.

Human oesophagus: a convenient antigenic substrate for the determination of anti-endomysium antibodies in the serological diagnosis of coeliac disease.

PubMed

Uibo, O; Lambrechts, A; Mascart-Lemone, F

1995-01-01

Immunoglobulin (Ig) A-class anti-endomysium antibodies are superior to other current antibody tests for detecting coeliac disease. We aimed to evaluate the suitability of human oesophagus for the determination of anti-endomysium antibodies. The specificity of monkey and human oesophageal tissue as antigenic substrate were compared using indirect immunofluorescence analysis. Overall, 159 individuals were studied: 56 patients with biopsy-proven coeliac disease (39 with active disease) and 103 controls. The patients' IgA-class anti-endomysium antibodies were compared using unfixed cryostat sections of human and monkey oesophagus. Indirect immunofluorescence analysis was performed with an initial serum sample dilution of 1:5, and if positive, the highest dilution yielding a positive reaction was reported. The anti-endomysium antibody test was positive in 38 out of 39 patients with active coeliac disease using monkey oesophagus (sensitivity 97%) and in all 39 patients with active coeliac disease using human oesophagus (sensitivity 100%). Ten out of 17 coeliac patients on a gluten-free diet had positive anti-endomysium antibodies using monkey oesophagus and 12 using human oesophagus as the antigenic substrate. This test was negative in all 103 controls using both substrates. Our study shows that human oesophageal tissue can be used instead of monkey tissue for determining anti-endomysium antibodies. Human tissue is a more sensitive antigenic substrate than monkey oesophagus and can be used to determine low titres of antibodies. Improving the diagnostic sensitivity of the anti-endomysium antibody test would make an important contribution to screening for coeliac disease.

Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

PubMed

Chithiwala, Zahabiya H; Lee, Hoi Chang; Hill, David L; Jellerette-Nolan, Teru; Fissore, Rafael; Grow, Daniel; Dumesic, Daniel A

2015-09-01

The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.

2008-07-15

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cellsmore » treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.« less

ICARE improves antinuclear antibody detection by overcoming the barriers preventing accreditation.

PubMed

Bertin, Daniel; Mouhajir, Yassin; Bongrand, Pierre; Bardin, Nathalie

2016-02-15

Antinuclear antibodies (ANA) are useful biomarkers for the diagnosis and the monitoring of rheumatic diseases. The American College of Rheumatology has stated that indirect immunofluorescence (IIF) analysis remains the gold standard for ANA screening. However, IIF is time consuming, subjective, not fully standardized and presents several issues for accreditation which is the process leading to ISO 15189 certification for medical laboratories. We propose an innovative tool for accreditation by using the quantitative evaluation of the automated image capture and analysis "ICARE" (Immunofluorescence for Computed Antinuclear antibody Rational Evaluation). We established the optimal screening dilution (1:160) and a fluorescence index (FI) cutoff for ICARE on a cohort of 91 healthy blood donors. Then, we evaluated performance of ICARE on a routine cohort of 236 patients. Precision parameters of ANA detection by IIF were evaluated according to ISO 15189. ICARE showed an excellent concordance with visual evaluation (88%, Kappa=0.76) and significantly discriminated between weak to moderate (1:160-1:320 titers) and high (>1:320 titers) ANA levels. A significant correlation was found between FI and ANA titers (Spearman's ρ=0.67; P<0.0001). Using ICARE, we reported precision parameters such as repeatability (CV<13.8%) and reproducibility (CV<13.1%) as well as absence of inter-sample contamination for ANA detection by IIF according to ISO 15189 standards. ICARE offers a precious help for the accreditation of IIF qualitative methods. This innovative quantitative approach is in adequacy with the process of continuous improvement of the quality of clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

A High Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains (PODs)

PubMed Central

Yip, Kenneth W.; Cuddy, Michael; Pinilla, Clemencia; Giulanotti, Marc; Heynen-Genel, Susanne; Matsuzawa, Shu-ichi; Reed, John C.

2014-01-01

PML is a tumor suppressor that promotes apoptosis through both p53-dependent and - independent mechanisms, participates in Rb-mediated cell cycle arrest, inhibits neoangiogenesis, and contributes to maintenance of genomic stability. PML also plays a role in host defense against viruses, conferring antiviral activity. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells, thus providing a morphological indicator. Known activators of PML include arsenicals and interferons, however, these agents induce a plethora of toxic effects, limiting their effectiveness. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML. We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may restore the tumor suppressor activity of PML in cancers or possibly promote anti-viral states. PMID:21233309

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

PubMed

Galay, Remil Linggatong; Matsuo, Tomohide; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Kusakisako, Kodai; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2018-04-01

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Pituitary Antibodies by Immunofluorescence: Approach and Results in Patients With Pituitary Diseases

PubMed Central

Ricciuti, Adriana; De Remigis, Alessandra; Landek-Salgado, Melissa A.; De Vincentiis, Ludovica; Guaraldi, Federica; Lupi, Isabella; Iwama, Shintaro; Wand, Gary S.; Salvatori, Roberto

2014-01-01

Context: Pituitary antibodies have been measured mainly to identify patients whose disease is caused or sustained by pituitary-specific autoimmunity. Although reported in over 100 publications, they have yielded variable results and are thus considered of limited clinical utility. Objectives: Our objectives were to analyze all publications reporting pituitary antibodies by immunofluorescence for detecting the major sources of variability, to experimentally test these sources and devise an optimized immunofluorescence protocol, and to assess prevalence and significance of pituitary antibodies in patients with pituitary diseases. Study Design and Outcome Measures: We first evaluated the effect of pituitary gland species, section fixation, autofluorescence quenching, blockade of unwanted antibody binding, and use of purified IgG on the performance of this antibody assay. We then measured cross-sectionally the prevalence of pituitary antibodies in 390 pituitary cases and 60 healthy controls, expressing results as present or absent and according to the (granular, diffuse, perinuclear, or mixed) staining pattern. Results: Human pituitary was the best substrate to detect pituitary antibodies and yielded an optimal signal-to-noise ratio when treated with Sudan black B to reduce autofluorescence. Pituitary antibodies were more common in cases (95 of 390, 24%) than controls (3 of 60, 5%, P = .001) but did not discriminate among pituitary diseases when reported dichotomously. However, when expressed according to their cytosolic staining, a granular pattern was highly predictive of pituitary autoimmunity (P < .0001). Conclusion: We report a comprehensive study of pituitary antibodies by immunofluorescence and provide a method and an interpretation scheme that should be useful for identifying and monitoring patients with pituitary autoimmunity. PMID:24606106

The value of tests of cure following cervical chlamydial infection.

PubMed

White, D J; Mann, C H; Matthews, R S; Leeming, J G; Clay, J C

1993-01-01

Test of cure (TOC) was performed 2, 4 and 6 weeks after treatment for cervical chlamydia infection with 10-14 days of Deteclo one tablet twice daily, erythromycin 500 mg twice daily or doxycycline 100 mg twice daily. Testing was by chlamydia culture and IDEIA (DAKO diagnostics Ltd). Discrepant results were subsequently checked by immunofluorescence (Syva MicroTrak) of both sets of left over transport media. Two hundred and three patients attended on at least one occasion; 189, 146 and 107 at 2, 4 and 6 weeks respectively. Of these 127, 70 and 34, respectively, denied sexual intercourse or had consistently used condoms. Fourteen were positive over the study period by either or both methods of detection. Of 8 culture positive results 3 were negative by IDEIA. Two of these had elementary bodies (EBs) on immunofluorescence of both sets of saved transport media. One had EBs on immunofluorescence of the saved culture transport medium only. None of the 6 IDEIA positive, culture negative patients had immunofluorescent EBs in the IDEIA transport media although one had EBs in the saved culture transport medium. One IDEIA suspicious, culture negative patient had EBs in both sets of saved transport media. There was no significant difference in the rate of chlamydia detection from patients admitting to or denying unprotected intercourse. TOC has a low yield in cases of cervical chlamydial infection when there has been careful contact tracing and treatment has been completed. If TOC is performed culture should be used if available and where antigen detection methods are used confirmation should be sought for any positive results.

Coordinated induction of cell survival signaling in the inflamed microenvironment of the prostate.

PubMed

McIlwain, David W; Zoetemelk, Marloes; Myers, Jason D; Edwards, Marshé T; Snider, Brandy M; Jerde, Travis J

2016-06-01

Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation. We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3). Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens. The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation. © 2016 Wiley Periodicals, Inc.

Risk factors for technical failure of endoscopic double self-expandable metallic stent placement by partial stent-in-stent method.

PubMed

Kawakubo, Kazumichi; Kawakami, Hiroshi; Toyokawa, Yoshihide; Otani, Koichi; Kuwatani, Masaki; Abe, Yoko; Kawahata, Shuhei; Kubo, Kimitoshi; Kubota, Yoshimasa; Sakamoto, Naoya

2015-01-01

Endoscopic double self-expandable metallic stent (SEMS) placement by the partial stent-in-stent (PSIS) method has been reported to be useful for the management of unresectable hilar malignant biliary obstruction. However, it is technically challenging, and the optimal SEMS for the procedure remains unknown. The aim of this study was to identify the risk factors for technical failure of endoscopic double SEMS placement for unresectable malignant hilar biliary obstruction (MHBO). Between December 2009 and May 2013, 50 consecutive patients with MHBO underwent endoscopic double SEMS placement by the PSIS method. We retrospectively evaluated the rate of successful double SEMS placement and identified the risk factors for technical failure. The technical success rate for double SEMS placement was 82.0% (95% confidence interval [CI]: 69.2-90.2). On univariate analysis, the rate of technical failure was high in patients with metastatic disease and unilateral placement. Multivariate analysis revealed that metastatic disease was a significant risk factor for technical failure (odds ratio: 9.63, 95% CI: 1.11-105.5). The subgroup analysis after double guidewire insertion showed that the rate of technical success was higher in the laser-cut type SEMS with a large mesh and thick delivery system than in the braided type SEMS with a small mesh and thick delivery system. Metastatic disease was a significant risk factor for technical failure of double SEMS placement for unresectable MHBO. The laser-cut type SEMS with a large mesh and thin delivery system might be preferable for the PSIS procedure. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Double versus single stenting for coronary bifurcation lesions: a meta-analysis.

PubMed

Katritsis, Demosthenes G; Siontis, George C M; Ioannidis, John P A

2009-10-01

Several trials have addressed whether bifurcation lesions require stenting of both the main vessel and side branch, but uncertainty remains on the benefits of such double versus single stenting of the main vessel only. We have conducted a meta-analysis of randomized trials including patients with coronary bifurcation lesions who were randomly selected to undergo percutaneous coronary intervention by either double or single stenting. Six studies (n=1642 patients) were eligible. There was increased risk of myocardial infarction with double stenting (risk ratio, 1.78; P=0.001 by fixed effects; risk ratio, 1.49 with Bayesian meta-analysis). The summary point estimate suggested also an increased risk of stent thrombosis with double stenting, but the difference was not nominally significant given the sparse data (risk ratio, 1.85; P=0.19). No obvious difference was seen for death (risk ratio, 0.81; P=0.66) and target lesion revascularization (risk ratio, 1.09; P=0.67). Stenting of both the main vessel and side branch in bifurcation lesions may increase myocardial infarction and stent thrombosis risk compared with stenting of the main vessel only.

GACD: Integrated Software for Genetic Analysis in Clonal F1 and Double Cross Populations.

PubMed

Zhang, Luyan; Meng, Lei; Wu, Wencheng; Wang, Jiankang

2015-01-01

Clonal species are common among plants. Clonal F1 progenies are derived from the hybridization between 2 heterozygous clones. In self- and cross-pollinated species, double crosses can be made from 4 inbred lines. A clonal F1 population can be viewed as a double cross population when the linkage phase is determined. The software package GACD (Genetic Analysis of Clonal F1 and Double cross) is freely available public software, capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in clonal F1 and double cross populations. Three functionalities are integrated in GACD version 1.0: binning of redundant markers (BIN); linkage map construction (CDM); and QTL mapping (CDQ). Output of BIN can be directly used as input of CDM. After adding the phenotypic data, the output of CDM can be used as input of CDQ. Thus, GACD acts as a pipeline for genetic analysis. GACD and example datasets are freely available from www.isbreeding.net. © The American Genetic Association. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Qualitative Examination of Children's Naming Skills through Test Adaptations.

ERIC Educational Resources Information Center

Fried-Oken, Melanie

1987-01-01

The Double Administration Naming Technique assists clinicians in obtaining qualitative information about a client's visual confrontation naming skills through administration of a standard naming test; readministration of the same test; identification of single and double errors; cuing for double naming errors; and qualitative analysis of naming…