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At least 1,189 records · Page 66Linked to original sources
PubMed2001

nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BK(Ca)) channels were located in discrete peripheral sites, some with Cav. Immunoreactivities not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BK(Ca) channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.

Animals↗
PubMed2006

Inorganic phosphate nanorods are a novel fluorescent label in cell biology.

We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO4.H2O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 microg/ml, the use of [3H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes.

Journal Article↗
PubMed2006

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice.

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259-273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259-273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.

Amiloride↗
PubMed2002

[Long-term outcome of gfp gene modified human RPE xenografts into the subretinal space of rabbits].

The possibility of delivery genes to retina by using retinal pigment epithelium (RPE) transplantation was investigated. Cultured human adult RPE cells were transfected with retrovirus encoding green fluorescent protein (GFP), then GFP-expressing RPE cells were transplanted into the subretinal space of rabbits through pars plana vitrectomy under microscopy. The transplant sites were examined in vivo by ophthalmoscope for fluorescence. The animals were euthanatized at 1, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 33, 54 weeks, respectively, and the retinas were studied histologically, including epi-fluorescent microscopy, confocal microscopy and transmission electron microscopy. The GFP-expressing transplant could be followed in the living retina up to 21 days by ophthalmoscope. The epi-fluorescent microscopy examination disclosed survival of the transplanted cells in 1 year with continual and significant GFP expression. It was observed the transplanted hRPE-gfp cells were not only present at transplant sites but spread over two to three quarters of retina. Generally, RPE xenografts integrated individually into host RPE in a regular cobblestones arrangement or formed a monolayer sheet between host RPE and neural retina. It was observed that intravitreal injection of FK-506 weekly improved the survival of RPE xenografts and reduced the infiltration of mononuclear microphages and lymphocytes in choroids from 1 through 14 weeks after transplantation. The results demonstrate a long-term survival of gene modified human RPE xenografts into the subretinal space of rabbits and support the feasibility of this approach for delivery genes to retina.

Adult↗
PubMed2003

[Arsenic trioxide inhibits P-glycoprotein expression in multidrug-resistant human leukemia K562/ADM cell line that overexpresses mdr-1 gene and enhances their chemotherapeutic sensitivity].

OBJECTIVE: To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and P-glyco-protein (P-gp) expression of multidrug-resistant human leukemia K562/ADM cells, and the combined effects of As(2)O(3) with conventional chemotherapeutic agents. METHODS: Multidrug-resistant human leukemia cell line K562/ADM that overexpresses mdr-1 gene was used as the target cells. The cell proliferating activity was assessed with a MTT assay. Cell morphology was examined by light microscopy, confocal microscopy and electron-microscopy. P-gp expression, cell-cycle status were determined by flow cytometry. RESULTS: K562/ADM cells were highly resistant to adriamycin, and cross-resistant to daunorubicin and etoposide. As(2)O(3) at concentrations of 0.5 to 20 micromol/L inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than their parent K562 cells did. As(2)O(3) induced marked apoptosis of K562/ADM cells showed by typical apoptotic morphological changes and the appearance of high sub-G(1) cell population. As(2)O(3) significantly inhibited the P-gp expression in K562/ADM cells, and exerted a synergistic effect on the enhancement of the cell sensitivity to adriamycin, daunorubicin and etoposide. CONCLUSION: As(2)O(3) induces growth-inhibition and apoptosis of multidrug-resistant K562/ADM cells, and augments synergistically the sensitivity of the cells to conventional chemotherapeutic agents via down-regulation of P-gp expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Inhibitory effects of alpha-interferon on epidermal growth factor-mediated receptor-dependent events.

To examine the mechanisms by which alpha-interferon (IFN-alpha) inhibits growth factor-mediated proliferative responses, we examined specific ligand-activated, receptor-dependent events. In direct ligand binding studies, we showed that IFN-alpha treatment of cells leads to a reduction in epidermal growth factor (EGF) receptor recognition at the cell surface, coupled with an alteration in the binding characteristics of EGF for its specific receptors. Specifically, the heterogeneity of binding exhibited by EGF was affected, and there was loss of the high affinity binding component. EGF-induced autophosphorylation of the EGF receptor was unaffected by IFN treatment. The trafficking of EGF-receptor complexes was followed using three-dimensional confocal microscopy. Confocal imaging revealed that the rapid internalization of EGF-receptor complexes was significantly reduced when cells were exposed to IFN. Accompanying the IFN-induced changes in receptor binding characteristics, we identified an alteration in EGF receptor gene expression; when cells were treated with IFN-alpha, elevated RNA levels specific for the EGF receptor were detected. Overall, IFN-alpha treatment inhibited EGF-induced cell proliferation. Our results imply that EGF-bound receptors that are unable to internalize are not fully competent with respect to signal regulation of both gene expression and growth. The data suggest that the signaling potential of the bound growth factor-receptor complex is apparently increased by an unspecified, species-specific, high affinity binding component. We propose that IFN treatment of responsive cell prevents the interaction of EGF-bound receptor with this component.

Blotting, Northern↗
PubMed1998

Antibodies against mesangial cells and their secretory products in chronic renal allograft rejection in the rat.

Antibodies or cell-mediated immunity can cause chronic rejection of vascularized organ grafts, but the nature and specificity of the antigen(s) involved has remained elusive. We have previously demonstrated the presence of antibodies against cryptic glomerular basement membrane antigens and undefined antigens in the mesangial area in rats with chronic renal allograft rejection. Current experiments were designed to study the post-transplant antibody response against cultured mesangial and endothelial cells in rats with chronic rejection using flow cytometry, indirect immunofluorescent staining, immunoelectron microscopy, confocal microscopy, and Western blots. The results were compared with those obtained with alloantisera raised by immunization with cultured mesangial cells. Post-transplant and post-immunization sera contained IgG antibodies against trypsinized mesangial cells detected by flow cytometry. Indirect immunofluorescent studies using mesangial cells grown on coverslips showed autoantibody binding to cytoplasmic granules in cultures early after plating whereas staining of later cultures showed antibody binding in an interrupted, web-like pattern on the outside of the cells. Immunoelectron microscopy showed autoantibody binding to intracellular secretory granules and to cell surface focal adhesion plaques. The latter finding was confirmed in double-labeling experiments with an antiserum against vinculin. Western blots with mesangial cell culture supernatants demonstrated autoantibody reactivity with antigens in the 40-kd and 60- to 70-kd range, and immunoprecipitation identified these molecules as biglycan and decorin. Absorption of the sera with mesangial cell culture supernatant removed most of the antibodies except those that gave a punctate staining with the mesangial cell surface. However, not all immunostaining of mesangial cells could be explained by antibodies against biglycan and decorin. Post-transplant sera, furthermore, contained low-titered antibodies against endothelial cells. We conclude that rats with chronic renal transplant rejection produce a strong autoantibody response against mesangial cell focal adhesion plaques and proteins secreted by these cells in culture. Such antibodies may cause local damage and interfere in the tissue repair process after injury.

Animals↗
PubMed2005

[Confocal scanning microscopy for biomedicine].

Confocal scanning microscopy is a new technique which can obtain the 3-D images of samples by using scanning and computer processing. It has been widely used in biomedical fields owing to its advantages such as high resolution and 3-D imaging. In this paper, we analyze the principle and research development of confocal scanning microscopies, and illustrate its advantages by comparing it with other microscopies.

Image Enhancement↗
PubMed1989

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy.

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.

Animals↗
PubMedDate not supplied

The histopathology of kidney uriniferous tubules as revealed by noninvasive confocal vital microscopy.

Tandem scanning confocal microscopy (TSCM) permits the noninvasive microscopic viewing of unstained, living, solid organs in real time. This article provides an overview of recent studies conducted by the author and other scientists, using TSCM and demonstrating its advantages in evaluating the histopathology of uriniferous tubules associated with normothermic renal ischemia, the nephrotic syndrome, and extracorporeal cold-storage preservation of kidneys.

Animals↗
PubMed1991

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy.

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.

Antibodies↗
PubMed1998

Interface inflammation after laser in situ keratomileusis. Sands of the Sahara syndrome.

PURPOSE: To determine the source of the interface debris that causes the interface inflammation known as "sands of the Sahara" after laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans, USA. METHODS: A microkeratome (Automated Corneal Shaper) was used to make a LASIK flap in 8 eyes of 4 rabbits. In 4 eyes, the blade was used directly from the sterile pack; in the contralateral 4 eyes, the blade was cleaned prior to use. In vivo confocal microscopy of the corneas was performed 1 day after surgery. An unused, cleaned blade and an unused, uncleaned blade, as well as blades used in the rabbit eyes, were examined by scanning electron microscopy. RESULTS: Confocal microscopy revealed numerous fragments of debris surrounded by inflammatory cells in the LASIK flap interfaces created by blades taken directly from the sterile package. Interfaces created by the cleaned blades showed only rare, scattered bits of debris. Scanning electron microscopy of the unused blades showed debris on the uncleaned blade removed directly from the sterile package. CONCLUSION: Post-LASIK interface inflammation may be caused by debris on the microkeratome blade, although other sources are possible. The interface debris and inflammation can be reduced or eliminated by cleaning the microkeratome blade before use.

Animals↗
PubMed1995

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study.

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Animals↗
PubMed2005

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

Biochemistry↗
PubMed2003

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Animals↗