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Scanning Confocal Microscopy (scanning + confocal_microscopy)
Kinds of Scanning Confocal Microscopy Selected AbstractsIn Situ Characterization of a Nb and Mo Containing , -TiAl Based Alloy Using Neutron Diffraction and High-Temperature Microscopy,ADVANCED ENGINEERING MATERIALS, Issue 11 2009Ian J. Watson Abstract In recent times, novel titanium aluminides containing the bcc , -phase at high temperatures are being developed for improved hot-working capabilities, however, predictions of the phase diagrams are merely uncertain. Here we present in-situ neutron studies, which are particularly sensitive to the atomic disorder in the ordered phases. Complementary laser scanning confocal microscopy is employed for in-situ microstructural investigations. [source] Transgene excision in zebrafish using the phiC31 integraseGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2010James A. Lister Single optical section of a 72 hour post-fertilization T2K-XpGbR (EF1-alpha-attP-GFP-attB-DsRed-Express) transgenic zebrafish larva obtained by laser scanning confocal microscopy. Injection of phiC31 integrase messenger RNA at the one-cell stage induces recombination of the transgene in a mosaic fashion, resulting in excision of the green fluorescent protein cassette and expression of DsRed fluorescent protein. Here recombination is evident in the lens and neuromasts of the anterior lateral line. See the article by Lister in this issue. [source] Reorganization of the nervous system during metamorphosis of a hydrozoan planulaINVERTEBRATE BIOLOGY, Issue 3 2000Vicki J. Martina Abstract. Laser scanning confocal microscopy is used to reveal the changes that occur in the RFamide-positive nerve net as a free-swimming, solid hydrozoan planula larva is transformed into a sessile, hollow, young polyp. Seven stages of development in Pennaria tiarella are described: planula competent to metamorphose, attaching planula, disc, pawn, crown, developing polyp, and developed primary polyp. The RFamide-positive nervous system undergoes dramatic reorganization during metamorphosis: (1) larval neurons degenerate; (2) new neurons differentiate and reform a nerve net; and (3) the overall distribution pattern of the nervous system changes. This study confirms earlier observations on RFamide-positive neurons of Hydractinia which also show the loss of these cells after the onset of metamorphosis. [source] Bis(4,7-dimethyl and 5-dinitro-1,10-phenanthroline) sulfato-oxovanadium(IV)-mediated in vivo male germ cell apoptosisJOURNAL OF APPLIED TOXICOLOGY, Issue 4 2001Osmond J. D'Cruz Abstract Oxovanadium(IV) [VO] complexes of 1,10-phenanthroline are a new class of potent apoptosis-inducing cytotoxic agents against human testicular cancer cells in vitro. The present study investigated the in vivo ability of four(bis)-chelated 1,10-phenanthroline [phen] complexes of sulfato-oxovanadium(IV),VO(phen)2, VO(Cl,phen)2, VO(Me2,phen)2 and VO(NO2,phen)2,with and without substitutions, to induce testicular germ cell apoptosis. Male germ cell loss in mice was measured by determining the epididymal sperm count, testicular weight and histological evaluation of the testes. Repetitive intratesticular injection (7.5 mg kg,1 testis,1) of bis-chelated 1,10-phenanthroline complexes of oxovanadium(IV) with 4,7-dimethyl [VO(Me2,phen)2] and 5-dinitro [VO(NO2,phen)2] substitution led to decreased sperm counts and reduced testicular weights. Histopathological examination of testicular sections from VO(Me2,phen)2 - and VO(NO2,phen)2 -treated mice revealed a marked inhibition of spermatogenesis and preferential loss of maturing, as well as elongated spermatids. In situ evaluation of seminiferous tubule cross-sections by terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick end-labeling (TUNEL) and laser scanning confocal microscopy showed characteristic apoptotic germ cells delineating the periphery of the seminiferous tubules. The ability of bis-chelated 4,7-dimethyl- and 5-dinitro-substituted 1,10-phenanthroline complexes of oxovanadium(IV) to induce germ cell apoptosis in vivo may have potential utility in the treatment of human testicular germ cell tumors. Copyright © 2001 John Wiley & Sons, Ltd. [source] Expression and subcellular location of COX-2 in human gastric cancer cellsJOURNAL OF DIGESTIVE DISEASES, Issue 2 2001Li Ling OBJECTIVE: To detect the expression of cyclooxygenase (COX) in human gastric cancer cell lines and determine the subcellular location of its isoforms. METHODS: Immunohistochemistry, reverse transcription,polymerase chain reaction (RT-PCR), and laser scanning confocal microscopy (LSCM) were used to investigate the expression and distribution of COX. RESULTS: Positive staining for COX-2 and COX-1 protein was seen in human gastric cancer cell lines MKN45, SGC7901 and AGS. However, COX-2 staining was absent and COX-1 staining was weak in the MGC803 cell line, although COX-2 mRNA was present in all four cell lines. When compared with COX-1, COX-2 was more strongly expressed at both protein and mRNA levels in the gastric cancer cell lines, which was confirmed by double labeling and LSCM. A quantitative analysis of fluorescein intensity indicated that the pixel intensity peak of COX-2 had a gray scale value of 50,70, while COX-1 was only 10. The LSCM technique also revealed the presence of COX-2 in the cytoplasm and nuclear envelope and COX-1 in the cytoplasm only. CONCLUSIONS: In human gastric cancer cells, COX-2 is expressed at higher levels than COX-1 and the different distributions of the two isoforms suggest that their roles in cell function are distinct. [source] Probing platelet factor 4 ,-granule targetingJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2004V. Briquet-Laugier Summary., The storage mechanism of endogenous secretory proteins in megakaryocyte ,-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet ,-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in ,-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify ,-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in ,-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze ,-granule biogenesis and its alterations in the congenital defect gray platelet syndrome. [source] "Sponge-like" structures in polymer blends: visualization, physico-mathematical analysis, and universalityMACROMOLECULAR SYMPOSIA, Issue 1 2002Takeji Hashimoto Mesoscopic structures formed during an ordering process in thermodynamically unstable, isometric, binary molecular mixtures were explored by time-resolved scattering (TRS) and laser scanning confocal microscopy (LSCM). Three-dimensional (3D) bicontinuous structures, which were constructed for the first time by time-resolved LSCM, were found to have a "sponge-like" structure composed of two phases. The structure factor obtained by 3D Fourier transformation of the sponge was found to be identical to that obtained by TRS, confirming that the sponge truly reflects the structural entities evolving in the system. Furthermore, the sponge was shown for the first time to be theoretically predictable by using 3D computer simulations based on the time-dependent Ginzburg-Landau theory. The sponge was subjected to differential geometrical analysis: its Gaussian curvature K, mean curvature H, and their distributions were successfully determined for the first time. The result revealed that the sponge has hyperbolic interfaces with area-averaged curvatures satisfying Role of MMP9 on invadopodia formation in cells from adenoid cystic carcinoma.MICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2010Study by laser scanning confocal microscopy Abstract Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] ,Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis unifloraNEW PHYTOLOGIST, Issue 1 2006Laura Domínguez Summary ,,Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. ,,Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. ,,Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. ,,These propagules may detach from the roots, establishing new plants. [source] In vivo corneal confocal microscopy in keratoconusOPHTHALMIC AND PHYSIOLOGICAL OPTICS, Issue 3 2005Joanna G. Hollingsworth Abstract Purpose:, To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. Methods:, Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 ± 10 years; range 16,49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. Results:, Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t -test: t = 1.82, p = 0.08). Conclusions:, Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus. [source] Real-time Visualization of Photochemically Induced Fluorescence of 8-Halogenated Quinolones: Lomefloxacin, Clinafloxacin and Bay3118 in Live Human HaCaT Keratinocytes,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2010Edmond B. Koker Halogenoquinolones are potent and widely used antimicrobials blocking microbial DNA synthesis. However, they induce adverse photoresponses through the absorption of UV light, including phototoxicity and photocarcinogenicity. The phototoxic responses may be the result of photosensitization of singlet oxygen, production of free radicals and/or other reactive species resulting from photodehalogenation. Here, we report the use of laser scanning confocal microscopy to detect and to follow the fluorescence changes of one monohalogenated and three di-halogenated quinolones in live human epidermal keratinocyte cells during in situ irradiation by confocal laser in real time. Fluorescence image analysis and co-staining with the LysoTracker probe showed that lysosomes are a preferential site of drug localization and phototransformations. As the lysosomal environment is relatively acidic, we also determined how low pH may affect the dehalogenation and concomitant fluorescence. With continued UV irradiation, fluorescence increased in the photoproducts from BAY y3118 and clinafloxacin, whereas it decreased for lomefloxacin and moxifloxacin. Our images not only help to localize these phototoxic agents in the cell, but also provide means for dynamic monitoring of their phototransformations in the cellular environment. [source] Reactive compatibilization of biodegradable poly(lactic acid)/poly(,-caprolactone) blends with reactive processing agentsPOLYMER ENGINEERING & SCIENCE, Issue 7 2008Masaki Harada Poly(lactic acid) (PLA) blended with poly(,-caprolactone) (PCL) was prepared with various reactive processing agents. Four isocyanates-lysine triisocyanate (LTI); lysine diisocyanate (LDI); 1,3,5-tris(6-isocyanatohexyl)-1,3,5-triazinane-2,4,6-trione (Duranate TPA-100); 1,3,5-tris(6-isocyanatohexyl)biuret (Duranate 24A-100)-and an industrial epoxide-trimethylolpropane triglycidyl ether (Epiclon 725)-were used as reactive processing agents. PLA/PCL blended in the presence of LTI had the highest torque in a mixer test. The test specimens were prepared by injection molding. The mechanical properties, thermal properties, molecular weight, melt viscosity, phase behavior, and morphology were investigated using tensile strength, impact strength, differential scanning calorimetry, melt mass-flow rate measurements, capillary rheometery, gel permeation chromatography, laser scanning confocal microscopy (LSCM), and visco-elasticity atomic force microscopy (VE-AFM). The impact strength increased considerably at 20 wt% PCL. The nominal tensile strain of PLA/PCL blended with LTI increased by 270%. The MFR values of PLA/PCL blends decreased with increasing LTI. Similar results were observed for shear viscosity. LSCM measurements showed that the diameters of PCL were dispersed about 0.4 ,m in the presence of LTI. VE-AFM showed that spherical particles with diameters of 50 nm were PCL-rich domain. These results indicate that isocyanate groups of LTI react with both terminal hydroxyl or carboxyl groups of polymers, and the compatibility of PLA/PCL blends improves with LTI by reactive processing. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers [source] Characteristics and function of cardiac mitochondrial nitric oxide synthaseTHE JOURNAL OF PHYSIOLOGY, Issue 4 2009Elena N. Dedkova We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H2DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations ([Ca2+]i= 1, 2 and 5 ,m) resulted in a dose-dependent increase of NO production by mitochondria when l -arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of l -arginine, mitochondrial NO production during stimulation of Ca2+ uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit l -arginine availability resulted in 50% inhibition of Ca2+ -induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)- N -(4-amino-5[aminoethyl]aminopentyl)- N,-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor l - N5 -(1-iminoethyl)ornithine (l -NIO) or iNOS inhibitor N -(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca2+ -induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of l -arginine, mitochondrial Ca2+ uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by l -arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH4; 100 ,m) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca2+ uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l -arginine and BH4. In conclusion, our data show the importance of l -arginine and BH4 for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS. [source] Expression and function of ryanodine receptors in rabbit penile corpus cavernosum smooth muscle cellsANDROLOGIA, Issue 3 2009H. G. Liu Summary This study aimed to investigate the expression and function of ryanodine receptors (RyRs) in rabbit penile corpus cavernosum smooth muscle (CCSM) cells. New Zealand white rabbit CCSM cells were cultured by primary tissue culture and identified by immunofluorescence technique. mRNA of three RyRs subunits in cultured CCSM cells was detected by reverse transcription polymerase chain reaction (RT-PCR). After excitation by noradrenaline, the concentration of Ca2+ in CCSM cells was detected by laser scanning confocal microscopy. It was found that only the RyRs1 subunit is expressed in CCSM by RT-PCR. The CCSM cells were divided into two groups: Group A, CCSM cells + 10 ,mol l,1 noradrenaline and Group B, CCSM cells + 50 ,mol l,1 procaine 10 ,mol l,1 noradrenaline. Compared with the base level and the level after noradrenaline excitation, fluorescence intensity improved 44.10 ± 6.01% in the test group A (n = 8) and 32.92 ± 4.92% (n = 8) in the procaine control group B, respectively. There were statistically significant differences between group A and group B (P < 0.01). It is concluded that RyRs1 subunit is expressed in CCSM cells and may contribute to regulating the cytoplasmic Ca2+ level. [source] Soil-borne wheat mosaic virus inclusion bodies: structural, compositional and staining propertiesANNALS OF APPLIED BIOLOGY, Issue 2 2003L J LITTLEFIELD Summary Anatomy and cytochemistry of inclusion bodies induced by Soil-borne wheat mosaic virus infection were studied in roots and leaves to learn more about the nature of inclusions and their roles in pathogenesis. Acid Fuchsin, Giemsa stain, Toluidine Blue and Trypan Blue stains facilitated visualization of inclusion bodies. Combined, simultaneous staining with Acid Fuchsin and Toluidine Blue clearly differentiated inclusion bodies from host nuclei. The overall anatomy, composition and structure of virus inclusions in leaves and roots were generally similar, as shown by phase contrast, differential interference contrast, epifluorescence, laser scanning confocal and transmission electron microscopy. Both were often closely associated with host nuclei; both were comprised of intertwined masses of tubular material, presumably endoplasmic reticulum, and in which varied numbers and sizes of vacuolar cavities occurred. Leaf inclusions, however, were typically larger and more vacuolate than those in roots. Lipids were found to be significant constituents of both the tubular and vacuolar components of inclusions, indicated by positive staining with Nile Red and Sudan Black. Inclusion bodies in both leaves and roots lost their structural and compositional integrity, eventually becoming disorganized and devoid of clearly identifiable components as host tissue aged and symptom expression advanced. Significant results of this study include the first published examination of virus inclusion bodies in root tissue, the degree of structural detail of inclusion body anatomy revealed by laser scanning confocal microscopy and the presence of an extensive lipid component in virus inclusion bodies. [source] Platelet C4d is highly specific for systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 2 2006Jeannine S. Navratil Objective Complement-activation product C4d is deposited on normal erythrocytes, while abnormal levels have been observed on the surface of erythrocytes of patients with systemic lupus erythematosus (SLE). This study examines whether C4d also deposits on human platelet surfaces, and whether platelet-bound C4d may provide a biomarker for SLE. Methods We conducted a cross-sectional study of 105 patients with SLE, 115 patients with other diseases, and 100 healthy controls. Levels of C4d on the surface of platelets were examined by flow cytometry and scanning confocal microscopy. Statistical analyses were performed to determine the clinical variables associated with platelet C4d. Results Abnormal levels of platelet C4d were found to be highly specific for SLE. Platelet C4d was detected in 18% of patients with SLE, being 100% specific for a diagnosis of SLE compared with healthy controls and 98% specific for SLE compared with patients with other diseases (P < 0.0001). In addition, platelet C4d was significantly associated with positivity for lupus anticoagulant (P < 0.0001) and anticardiolipin antibodies of the IgG (P = 0.035) or the IgM (P = 0.016) isotype. Platelet C4d was also significantly associated with SLE disease activity according to the SLE Disease Activity Index (P = 0.039), low serum C4 (P = 0.046), an elevated erythrocyte sedimentation rate (P = 0.006), and abnormal levels of C4d on erythrocytes (P < 0.0001). Conclusion This observation suggests that platelet-bound C4d may be a useful biomarker for SLE and may be a clue to the pathogenic mechanisms responsible for the myriad thrombotic and vascular complications of lupus associated with antiphospholipid antibodies. [source] Effects of ionic strength on lysozyme uptake rates in cation exchangers.BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005I: Uptake in SP Sepharose FF Fluorescence scanning confocal microscopy was used in parallel with batch uptake and breakthrough measurements of transport rates to study the effect of ionic strength on the uptake of lysozyme into SP Sepharose FF. In all cases the adsorption isotherms were near-rectangular. As described previously, the intraparticle profiles changed from slow-moving self-sharpening fronts at low salt concentration, to fast-moving diffuse profiles at high salt concentration, and batch uptake rates correspondingly increased with increasing salt concentration. Shrinking core and homogeneous diffusion frameworks were used successfully to obtain effective diffusivities for the low salt and high salt conditions, respectively. The prediction of column breakthrough was generally good using these frameworks, except for low-salt uptake results. In those cases, the compressibility of the stationary phase coupled with the shrinking core behavior appears to reduce the mass transfer rates at particle-particle contacts, leading to shallower breakthrough curves. In contrast, the fast uptake rates at high ionic strength appear to reduce the importance of mass transfer limitations at the particle contacts, but the confocal results do show a flow rate dependence on the uptake profiles, suggesting that external mass transfer becomes more limiting at high ionic strength. These results show that the complexity of behavior observable at the microscopic scale is directly manifested at the column scale and provides a phenomenological basis to interpret and predict column breakthrough. In addition, the results provide heuristics for the optimization of chromatographic conditions. © 2005 Wiley Periodicals, Inc. [source] Rab5a overexpression promoting ovarian cancer cell proliferation may be associated with APPL1-related epidermal growth factor signaling pathwayCANCER SCIENCE, Issue 6 2010Zhen Zhao Rab5a is a regulatory guanosine triphosphatase that is associated with the transport and fusion of endocytic vesicles, and participates in regulation of intracellular signaling pathways embraced by cells to adapt to the specific environment. Rab5a is also correlated with lung, stomach, and hepatocellular carcinomas. Here, we detected Rab5a in paraffin-embedded samples of 20 ovarian cysts, 20 benign cystadenomas, and 39 ovarian cancers by immunohistochemistry, and observed that Rab5a expression was significantly higher in ovarian cancer (P = 0.0001). By setting up stable HO-8910 cell lines expressing Rab5a or dominant negative Rab5a (Rab5a:S34N), we found that Rab5a overexpression enhanced the cell growth by promoting G1 into S phase. In contrast, Rab5a:S34N inhibited this process. Additionally, APPL1 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif), a downstream effector of Rab5a, was also involved in promoting HO-8910 cell cycle progress. But this function was blocked by Rab5a:S34N. Laser scanning confocal microscopy represented the colocalization of APPL1 and Rab5a in the plasmolemma, which changed with the time of epidermal growth factor (EGF) stimulation. We also found APPL1 could transfer from the membranes into the nucleus where it interacted with NuRD/MeCP1 (the nucleosome remodeling and histone deacetylase multiprotein complex). NuRD is reported to be involved in the deacetylation of histone H3 and H4 to regulate nuclear transcription. So Rab5a promoted proliferation of ovarian cancer cells, which may be associated with the APPL1-related epidermal growth factor signaling pathway. (Cancer Sci 2010) [source] In vivo confocal microscopy of the bulbar conjunctivaCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 4 2009Nathan Efron PhD DSc Abstract Background:, The aim of this work is to develop a more complete qualitative and quantitative understanding of the in vivo histology of the human bulbar conjunctiva. Methods:, Laser scanning confocal microscopy (LSCM) was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 healthy human volunteer subjects. Results:, The superficial epithelial layer of the bulbar conjunctiva is seen as a mass of small cell nuclei. Cell borders are sometimes visible. The light grey borders of basal epithelial cells are clearly visible, but nuclei can not be seen. The conjunctival stroma is comprised of a dense meshwork of white fibres, through which traverse blood vessels containing cellular elements. Orifices at the epithelial surface may represent goblet cells that have opened and expelled their contents. Goblet cells are also observed in the deeper epithelial layers, as well as conjunctival microcysts and mature forms of Langerhans cells. The bulbar conjunctiva has a mean thickness of 32.9 ± 1.1 µm, and a superficial and basal epithelial cell density of 2212 ± 782 and 2368 ± 741 cells/mm2, respectively. Overall goblet and mature Langerhans cell densities are 111 ± 58 and 23 ± 25 cells/mm2, respectively. Conclusions:, LSCM is a powerful technique for studying the human bulbar conjunctiva in vivo and quantifying key aspects of cell morphology. The observations presented here may serve as a useful marker against which changes in conjunctival morphology due to disease, surgery, drug therapy or contact lens wear can be assessed. [source] DOPAMINE D2 RECEPTOR STIMULATION INHIBITS ANGIOTENSIN II-INDUCED HYPERTROPHY IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2009Hong Li SUMMARY 1Myocardial hypertrophy is a common pathological change that accompanies cardiovascular disease. Dopamine D2 receptors have been demonstrated in cardiovascular tissues. However, the pathophysiological involvement of D2 receptors in myocardial hypertrophy is unclear. Therefore, the effects of the D2 receptor agonist bromocriptine and the D2 receptor antagonist haloperidol on angiotensin (Ang) II- or endothelin (ET)-1-induced hypertrophy of cultured neonatal rat ventricular myocytes were investigated in the present study. 2Protein content and protein synthesis, determined by examining [3H]-leucine uptake, were used as estimates of cardiomyocyte hypertrophy. The expression of D2 receptor protein in neonatal rat ventricular myocytes was determined using western blotting. Changes in [Ca2+]i in cardiomyocytes were observed by laser scanning confocal microscopy. 3Angiotensin II and ET-1, both at 10 nmol/L, induced myocyte hypertrophy, as demonstrated by increased protein content and synthesis, [Ca2+]i levels, protein kinase C (PKC) activity and phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and mitogen-activated protein kinase (MAPK) p38 (p38). Concomitant treatment of cells with 10 nmol/L AngII plus 10 µmol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy, MAPK phosphorylation and PKC activity in the membrane, as well as [Ca2+]i signalling pathways, compared with the effects of AngII alone. In addition, 10 µmol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy induced by 10 nmol/L ET-1. However, pretreatment with haloperidol (10 µmol/L) had no significant effects on cardiomyocyte hypertrophy induced by either AngII or ET-1. 4In conclusion, D2 receptor stimulation inhibits AngII-induced hypertrophy of cultured neonatal rat ventricular myocytes via inhibition of MAPK, PKC and [Ca2+]i signalling pathways. [source]
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