To see the other types of publications on this topic, follow the link: Immuno fluorescence microscopy.
Journal articles on the topic 'Immuno fluorescence microscopy'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Immuno fluorescence microscopy.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Fisher, Phyllis J., William S. Wessels, Allan B. Dietz, and Franklyn G. Prendergast. "Immuno-Fluorescence Scanning Electron Microscopy of Biological Cells." Microscopy Today 18, no. 5 (August 24, 2010): 8–13. http://dx.doi.org/10.1017/s1551929510000805.
Full textAbstract:
Scanning electron microscopy (SEM) can produce striking three-dimensional images of biological cells and tissues with submicron resolution of surface morphology. Such cell surfaces are often complex blends of folds, extrusions, and pockets that may be necessary in the positioning of specific molecules within interaction range of each other. Thus, surface changes can have a spatial control over some molecular functions, and identification of select molecules at distinct morphological locations becomes critical to our understanding of total cell function.
2
McMahon, J. T., J. L. Myles, and R. R. Tubbs. "A Novel use of Fluorescence Microscopy in Evaluation of Renal Biopsies." Microscopy and Microanalysis 3, S2 (August 1997): 355–56. http://dx.doi.org/10.1017/s1431927600008667.
Full textAbstract:
Light, immuno, and electron microscopy are standard for evaluation of renal biopsies. For light microscopy (LM), special stains including PAS and Jones silver methenamine (JSM) are frequently used to enhance recognition of renal anomalies related to disease. We recently discovered that fluorescence microscopy (FM) extends the usefulness of frozen and paraffin sections stained with hematoxylin and eosin and may presage the findings of immuno and electron microscopy. With UV illumination and reflected light fluorescence excitation, standard histologie sections reveal pathologic glomerular and other renal changes not evident by conventional bright field microscopy and may obviate the need for additional special stains.For frozen sections, tissues mounted on gum tragacanth were snap frozen in isopentane chilled with liquid nitrogen and cryosectioned at a thickness of 4-6 μm. For paraffin sections, tissues were fixed in neutral buffered 10% formalin or in Hollande’s fixative (3.6% formalin, 40% picric acid, 25% copper acetate, 1.5% acetic acid) and sectioned at 2-4 μm.
3
Diaspro, Alberto, Giuseppe Chirico, and Maddalena Collini. "Two-photon fluorescence excitation and related techniques in biological microscopy." Quarterly Reviews of Biophysics 38, no. 2 (May 2005): 97–166. http://dx.doi.org/10.1017/s0033583505004129.
Full textAbstract:
1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.
4
Paupard, Marie-Christine, Agnes Miller, Barth Grant, David Hirsh, and David H. Hall. "Immuno-EM Localization of GFP-tagged Yolk Proteins in C. Elegans Using Microwave Fixation." Journal of Histochemistry & Cytochemistry 49, no. 8 (August 2001): 949–56. http://dx.doi.org/10.1177/002215540104900803.
Full textAbstract:
Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)
5
Lange, Felix, Paola Agüi-Gonzalez, Dietmar Riedel, Nhu T. N. Phan, Stefan Jakobs, and Silvio O. Rizzoli. "Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging." PLOS ONE 16, no. 5 (May 10, 2021): e0240768. http://dx.doi.org/10.1371/journal.pone.0240768.
Full textAbstract:
Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.
6
Lammert, Frank. "Cholesterol crystal binding of biliary immuno-globulin A: visualization by fluorescence light microscopy." World Journal of Gastroenterology 7, no. 2 (2001): 198. http://dx.doi.org/10.3748/wjg.v7.i2.198.
Full text
7
Karreman, Matthia A., Alexandra V. Agronskaia, Elly G. van Donselaar, Karin Vocking, Farzad Fereidouni, Bruno M. Humbel, C. Theo Verrips, Arie J. Verkleij, and Hans C. Gerritsen. "Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen." Journal of Structural Biology 180, no. 2 (November 2012): 382–86. http://dx.doi.org/10.1016/j.jsb.2012.09.002.
Full text
8
LIU, Miaomiao, Jun XIE, Hailing ZHENG, Yang ZHOU, Bing WANG, and Zhiwen HU. "Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy." Analytical Sciences 31, no. 12 (2015): 1317–23. http://dx.doi.org/10.2116/analsci.31.1317.
Full text
9
TRUJILLO, O., C. GRIFFIS, Y. LI, and M. SLAVIK. "A MACHINE VISION SYSTEM USING IMMUNO-FLUORESCENCE MICROSCOPY FOR RAPID RECOGNITION OF SALMONELLA TYPHIMURIUM." Journal of Rapid Methods and Automation in Microbiology 9, no. 2 (August 2001): 115–34. http://dx.doi.org/10.1111/j.1745-4581.2001.tb00234.x.
Full text
10
Kinoshita, Takaaki, Yosio Mori, Kazumi Hirano, Shinya Sugimoto, Ken-ichi Okuda, Shunsuke Matsumoto, Takeshi Namiki, et al. "Immuno-Electron Microscopy of Primary Cell Cultures from Genetically Modified Animals in Liquid by Atmospheric Scanning Electron Microscopy." Microscopy and Microanalysis 20, no. 2 (February 25, 2014): 469–83. http://dx.doi.org/10.1017/s1431927614000178.
Full textAbstract:
AbstractHigh-throughput immuno-electron microscopy is required to capture the protein–protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allowsin situcorrelative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild typeDrosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from anankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell’s plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.
11
Brown, Gaie, James Aitken, Helen W. McL Rixon, and Richard J. Sugrue. "Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells." Journal of General Virology 83, no. 3 (March 1, 2002): 611–21. http://dx.doi.org/10.1099/0022-1317-83-3-611.
Full textAbstract:
We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.
12
Milrot, Elad, Efi Makdasi, Boaz Politi, Tomer Israely, and Orly Laskar. "A Cell-Based Capture Assay for Rapid Virus Detection." Viruses 12, no. 10 (October 15, 2020): 1165. http://dx.doi.org/10.3390/v12101165.
Full textAbstract:
Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.
13
Ching, Reagan W., Kashif Ahmed, Paul C. Boutros, Linda Z. Penn, and David P. Bazett-Jones. "Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP." Journal of Cell Biology 201, no. 2 (April 15, 2013): 325–35. http://dx.doi.org/10.1083/jcb.201211097.
Full textAbstract:
Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations. Using promyelocytic leukemia nuclear bodies (PML NBs) as a model, we used immuno-TRAP to determine if specific genes localize within molecular dimensions with these bodies. Although we confirmed a TP53 gene–PML NB association, immuno-TRAP allowed us to uncover novel locus-PML NB associations, including the ABCA7 and TFF1 loci and, most surprisingly, the PML locus itself. These associations were cell type specific and reflected the cell’s physiological state. Combined with microarrays or deep sequencing, immuno-TRAP provides powerful opportunities for identifying gene locus associations with potentially any nuclear subcompartment.
14
Tanjong, RE, G. Ikomey, M. Masembe, and TN Akenji. "Comparison of immuno-fluorescence microscopy and optical microscopy after Giemsa staining in the diagnosis of malaria during pregnancy in Buea, Cameroon." International Journal of Biological and Chemical Sciences 8, no. 4 (January 15, 2015): 1604. http://dx.doi.org/10.4314/ijbcs.v8i4.22.
Full text
15
Hjelm, Anna, Bill Söderström, David Vikström, Wouter S. P. Jong, Joen Luirink, and Jan-Willem de Gier. "Autotransporter-Based Antigen Display in Bacterial Ghosts." Applied and Environmental Microbiology 81, no. 2 (November 14, 2014): 726–35. http://dx.doi.org/10.1128/aem.02733-14.
Full textAbstract:
ABSTRACTBacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered theEscherichia coliautotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using theMycobacterium tuberculosisvaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorateE. coliandSalmonellaghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E inE. coliandSalmonellacan be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.
16
Chabanas, Agnès, Jean‐Jacques Rambeaud, Hong-Hsu Huo, Daniel Seigneurin, and Jean-Jacques Lawrence. "Limits of Flow-Cytometry Histogram Analysis Methods to Assess Bladder Tumour Antigen Expression." Analytical Cellular Pathology 13, no. 1 (1997): 39–47. http://dx.doi.org/10.1155/1997/312514.
Full textAbstract:
Tumour‐associated antigens detected in cells obtained from bladder washings or tumours are useful markers in bladder cancer. Flow cytometry is commonly used to quantify immuno‐stained cells. A straightforward way to analyze data is to count the fluorescent cells above a threshold empirically determined on a control histogram representation. However, specific antigens expressed at highly variable rates give rise to wide range distributions in flow cytometry as illustrated when a mucin antigen for urinary bladder was titrated by M344 monoclonal antibody in urothelial cancer cells. We have evaluated several methods of background estimation and subtraction in order to determine the proportion of M344 Mab positive cells. These includethreshold setting(Histogram Shape Dependent (HSD) threshold developped in this study, 2% preset or 5% preset background),subtraction of the blankfrom the test histograms, andKolmogorov–Smirnov statistical test. The HSD method appeared to be a more reliable method for background estimation; however, in the case of very low antigen expression, where specific fluorescence histograms could hardly be distinguished from that of the background, fluorescence microscopy remained the only valid method, since it allowed the distinction between specific and non‐specific fluorescence on the basis of structural differences between the two.
17
Hess, Michael W., Iris M. Krainer, Przemyslaw A. Filipek, Barbara Witting, Karin Gutleben, Ilja Vietor, Heinz Zoller, et al. "Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations." Journal of Clinical Medicine 10, no. 9 (April 28, 2021): 1901. http://dx.doi.org/10.3390/jcm10091901.
Full textAbstract:
Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.
18
Eissa, Samia. "CYTO-EMBRYOLOGICAL CHANGES IN BOTH FERTILIZED AND ARTIFICIALLY ACTIVATED SEA URCHIN EGG AS REVEALED BY IMMUNO-FLUORESCENCE MICROSCOPY." Egyptian Journal of Aquatic Biology and Fisheries 3, no. 3 (June 1, 1999): 43–62. http://dx.doi.org/10.21608/ejabf.1999.3425.
Full text
19
Struckman, Heather L., Stephen Baine, Justin Thomas, Louisa Mezache, Kirk Mykytyn, Sándor Györke, Przemysław B. Radwański, and Rengasayee Veeraraghavan. "Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle." Microscopy and Microanalysis 26, no. 1 (January 14, 2020): 157–65. http://dx.doi.org/10.1017/s1431927619015289.
Full textAbstract:
AbstractThe voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.
20
Pegon, Julie N., Cécile V. Denis, Soline Odouard, Olivier D. Christophe, and Peter J. Lenting. "Siglecs as Novel Cellular Partners for Von Willebrand Factor." Blood 116, no. 21 (November 19, 2010): 4306. http://dx.doi.org/10.1182/blood.v116.21.4306.4306.
Full textAbstract:
Abstract Abstract 4306 Introduction: Von Willebrand factor (VWF) is a multimeric protein that is pertinent to the haemostatic process by promoting platelet recruitment to the growing thrombus and acting as a carrier-protein for factor VIII. It is well established that VWF is able to interact with several cellular receptors present on the surface of platelets and leukocytes. In search for novel cellular partners for VWF present on these cells, we made use of the notion that VWF is heavily glycosylated, with the mature molecule containing 12 N-linked glycans and 10 O-linked glycans. Importantly, the vast majority of these glycans (>90%) are sialylated. The human proteome contains a family of sialic acid-binding receptors, known as Sialic acid-binding Immunoglobulin-like Lectins (Siglecs), which are expressed on cells of hematopoietic origin. The current study was designed to investigate the potential of Siglecs to interact with VWF. Methods: Stable human HEK293 cell lines were established that express human Siglec-5, Siglec-7 or Siglec-9. In addition, soluble variants of Siglecs were obtained: monomeric HPC4-tagged Siglec-5 and Siglec-7 (sSg5/HPC4 & sSg7/HPC4) and dimeric Fc-fusion proteins for human Siglec-3, -5, -7, -9, -10, -11 and murine Siglec-F (sSg-3/Fc etc). These Siglecs were used to study their interaction with purified plasma-derived (pdVWF) or recombinant BHK-cell derived VWF (rVWF). Protein-protein interactions were investigated via immuno-sorbent assays and via surface plasmon resonance (SPR) using Octet QK equipment. Binding of VWF to Siglec-expressing cells was assessed via immuno-fluorescence microscopy. Results: We observed consistent association of VWF (irrespective whether immuno-sorbent or SPR assays were used) to all of the immobilized Siglecs tested, with the exception of sSg-11/Fc that did not interact with pdVWF or with rVWF. Inversely, all soluble Siglecs but sSg-11/Fc efficiently bound to immobilized pdVWF or rVWF. Half-maximal binding in immuno-sorbent assays was between 97 and 645 nM (binding of VWF to immobilized Siglecs) versus 166 and 270 nM (binding of Siglecs to immobilized VWF). More detailed studies on the determination of the kinetic parameters using SPR technology are in progress. Specificity of the interaction was confirmed by treating VWF with sialidase before testing Siglec binding, and such treatment resulted in a strongly reduced (up to 80%) ability of Siglecs to bind to immobilized VWF. VWF was further found to bind to Siglec-5, -7 or -9 expressing HEK293 cells as assessed via immuno-fluorescence microscopy. In general, 10–15 % of the Siglec-expressing cells were covered with VWF after incubation, with the fluorescent intensity increasing dose-dependently with the applied VWF concentration. No fluorescent signal was observed upon incubation of VWF with non-transfected cells or when VWF was omitted from the incubation. Since Siglecs may be masked by the presence of endogenous sialylated proteins at the cellular surface, we also tested VWF binding to these cells following sialidase treatment. Sialidase treatment of cells resulted in a marked increase in the number of VWF-binding cells (up to 80% of the Siglec-positive cells) as well as an increase in intensity of the fluorescent signal. Co-localization of VWF with Siglecs at the cellular surface was confirmed by DuoLink-analysis, which allows the detection of proteins that are in the vicinity of ≤40 nm. Emerging evidence demonstrates that endothelial. Conclusions: We identified one murine and six human Siglec-family members as novel partners for VWF. Interactions between VWF and Siglecs are mediated by sialic acid structures present on VWF. In addition, the cellular accessibility of Siglecs for VWF is modulated by cis-acting sialylated proteins at the cell surface. In conclusion, our data demonstrate that the VWF glycome allows the interaction with a novel family of cellular receptors, potentially opening previously unrecognized avenues in the biology of VWF. Disclosures: No relevant conflicts of interest to declare.
21
Goddard, R. H., and J. W. La Claire II. "Organization and composition of the cytoskeleton of giant algal cells: Its role in wound healing." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 752–53. http://dx.doi.org/10.1017/s0424820100155736.
Full textAbstract:
We have been using the giant algal cells of Ernodesmis verticillata as a model system to study the process of wound healing at the cellular level of organization. Using immuno-localization techniques we have observed changes in the distribution of tubulin-containing microtubules (MTs) and actin-containing microfilaments (MFs), that occur during wound healing. Based on these and new findings, we carried out correlative experiments with inhibitors to investigate the functional roles of the various cytoskeletal components in wound healing.Emodesmis cells were cultured, Wounded and Fixed as described previously. The cytoplasm from fixed cells was adhered to polylysine-coated coverslips for light microscopy, or to formvar coated gold grids for transmission electron microscopy (TEM).The attached cells were labeled with primary antibodies (against tubulin ,actin,calmodulin [CAM], intermediate filament [If] proteins, or myosin)followed by incubation in appropriate secondary antibodies conjugated with FITC or TRITC for fluorescence, or conjugated to gold beads for TEM.
22
van der Loos, C. M., and A. E. Becker. "Double epi-illumination microscopy with separate visualization of two antigens: a combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining." Journal of Histochemistry & Cytochemistry 42, no. 3 (March 1994): 289–95. http://dx.doi.org/10.1177/42.3.7508469.
Full textAbstract:
We present a method for an epi-illumination immunohistochemical double staining approach. The method combines the use of an immuno-alkaline phosphatase technique and the immunogold-silver technique, visualized with epifluorescence and epi-polarization illumination, respectively. Out of six tested alkaline phosphatase activity-revealing methods, only the reaction product obtained with the Becton Dickinson CAS Red kit showed an intense red fluorescence with a rhodamine filter set and no signal with epi-polarization illumination. The silver precipitate did not exhibit any signal with the rhodamine filter set. This allows separate observation and photographic recording of two antigens in one tissue section, an objective that cannot be achieved with conventional immunoenzyme double staining methods. The double epi-illumination approach presented is compatible with different immunoenzyme double staining protocols.
23
Henson, John H., Bakary Samasa, Charles B. Shuster, and Athula H. Wikramanayake. "The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo." PLOS ONE 16, no. 5 (May 26, 2021): e0248197. http://dx.doi.org/10.1371/journal.pone.0248197.
Full textAbstract:
Canonical Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.
24
Gomez, F., P. Ruiz, J. A. Bernal, M. Escobar, A. Garcia-Egido, and J. J. B. Lopez-Saez. "Enhancement of Splenic-Macrophage Fcγ Receptor Expression by Treatment with Estrogens." Clinical Diagnostic Laboratory Immunology 8, no. 4 (July 1, 2001): 806–10. http://dx.doi.org/10.1128/cdli.8.4.806-810.2001.
Full textAbstract:
ABSTRACT Splenic-macrophage Fcγ receptors (FcγRs) participate in the pathophysiologies of immune-complex diseases and in host defense against infection. Modulation of macrophage FcγR expression is an immuno-therapeutic target. Glucocorticoids, sex steroids, and dopaminergic drugs modulate macrophage FcγR expression. Previous data indicate that estradiol increases macrophage FcγR expression. Nevertheless, the effects of clinically used estrogens upon macrophage FcγR expression are unknown. We assessed the effects of treatment with commonly used estrogens on the expression of macrophage FcγRs using a guinea pig experimental model. Six estrogens have been studied: ethynylestradiol (Et), mestranol (M), chlortianisene (Ct), promestriene, 17-epiestriol, and 17β-estradiol. Following in vivo treatment of guinea pigs, we determined the clearance of immunoglobulin G (IgG)-sensitized erythrocytes in vivo, the binding of IgG-sensitized erythrocytes by isolated splenic macrophages, and splenic-macrophage FcγR cell surface expression. Estrogens enhance the clearance of IgG-sensitized erythrocytes by increasing splenic-macrophage FcγR expression. Et, M, and Ct were more effective than the other estrogens. Flow cytometry and fluorescence microscopy with monoclonal antibodies demonstrated that estrogens increase the cell surface expression of FcγR1 and -2 more than that of FcγR2. These data indicate that treatment with commonly used estrogens enhances the clearance of IgG-sensitized cells by improving splenic-macrophage FcγR expression.
25
Zayas-Santiago, Astrid, David S. Ríos, Lidia V. Zueva, and Mikhail Y. Inyushin. "Localization of αA-Crystallin in Rat Retinal Müller Glial Cells and Photoreceptors." Microscopy and Microanalysis 24, no. 5 (September 26, 2018): 545–52. http://dx.doi.org/10.1017/s1431927618015118.
Full textAbstract:
AbstractTransparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as “living light guides” as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.
26
Wang, Xin-Ping, Bernd Walkenfort, Matthias König, Lisa König, Sabine Kasimir-Bauer, and Sebastian Schlücker. "Fast and reproducible iSERS microscopy of single HER2-positive breast cancer cells using gold nanostars as SERS nanotags." Faraday Discussions 205 (2017): 377–86. http://dx.doi.org/10.1039/c7fd00135e.
Full textAbstract:
Speed is often a bottleneck in conventional Raman microscopy on biological specimens. In immuno-Raman microspectroscopy, or for short iSERS microscopy, the acquisition times per pixel have been reduced by more than one order of magnitude over the past decade since its proof of concept. Typically rather high laser power densities are employed with the intention of compensating for the shorter acquisition times, without checking the reproducibility of the results in repeated experiments on the same sample. Here, we systematically analyze this aspect at the single-cell level since it forms the basis of quantification and is very important for reinspection of the same specimen. Specifically, we investigate experimentally the role of the laser power density in conjunction with the acquisition times per pixel in a series of repeated iSERS experiments on the same single cell overexpressing the breast cancer tumor marker human epidermal growth factor receptor 2 (HER2). Confocal iSERS mapping experiments were guided by wide-field fluorescence microscopy for selecting the regions of interest. We demonstrate that the combination of ca. a 1–2 mW laser power (40× objective, NA 0.6), 50 ms acquisition time per pixel and a high EM-CCD signal gain yields highly reproducible iSERS images in a series of four repeated experiments on the same single cell. In contrast, longer acquisition times (0.8 s, no EM gain) and in particular higher laser power (4 mW up to 18 mW) densities lead to non-reproducible iSERS results due to signal degradation.
27
Cuppen, E., M. Wijers, J. Schepens, J. Fransen, B. Wieringa, and W. Hendriks. "A FERM domain governs apical confinement of PTP-BL in epithelial cells." Journal of Cell Science 112, no. 19 (October 1, 1999): 3299–308. http://dx.doi.org/10.1242/jcs.112.19.3299.
Full textAbstract:
PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins. Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells. Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10–15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin. However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays. Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family.
28
Misevic, Gradimir, Iacob Checiu, and Octavian Popescu. "Glyconectin Cell Adhesion Epitope, β-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos." Molecules 26, no. 13 (June 30, 2021): 4012. http://dx.doi.org/10.3390/molecules26134012.
Full textAbstract:
Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan–glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 β-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan–glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.
29
Conese, M., A. Nykjaer, C. M. Petersen, O. Cremona, R. Pardi, P. A. Andreasen, J. Gliemann, E. I. Christensen, and F. Blasi. "alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor." Journal of Cell Biology 131, no. 6 (December 15, 1995): 1609–22. http://dx.doi.org/10.1083/jcb.131.6.1609.
Full textAbstract:
The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.
30
Fang, Lei, Xinggang Wang, Qingzhu Sun, Eleni Papakonstantinou, Chongteck S’ng, Michael Tamm, Daiana Stolz, and Michael Roth. "IgE Downregulates PTEN through MicroRNA-21-5p and Stimulates Airway Smooth Muscle Cell Remodeling." International Journal of Molecular Sciences 20, no. 4 (February 18, 2019): 875. http://dx.doi.org/10.3390/ijms20040875.
Full textAbstract:
The patho-mechanism leading to airway wall remodeling in allergic asthma is not well understood and remodeling is resistant to therapies. This study assessed the effect of immunoglobulin E (IgE) in the absence of allergens on human primary airway smooth muscle cell (ASMC) remodeling in vitro. ASMCs were obtained from five allergic asthma patients and five controls. Proliferation was determined by direct cell counts, mitochondrial activity by expression of cytochrome c, protein expression by immunoblotting and immuno-fluorescence, cell migration by microscopy imaging, and collagen deposition by cell based ELISA and RNA expression by real time PCR. Non-immune IgE activated two signaling pathways: (i) signal transducer and activator of transcription 3 (STAT3)→miR-21-5p→downregulating phosphatase and tensin homolog (PTEN) expression, and (ii) phosphatidylinositol 3-kinases (PI3K)→protein kinase B (Akt)→mammalian target of rapamycin (mTOR)→ribosomal protein S6 kinase beta-1 (p70s6k)→peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α)→peroxisome proliferator-activated receptor-γ (PPAR-γ)→cyclooxygenase-2 (COX-2)→mitochondrial activity, proliferation, migration, and extracellular matrix deposition. Reduced PTEN expression correlated with enhanced PI3K signaling, which upregulated ASMC remodeling. The inhibition of microRNA-21-5p increased PTEN and reduced mTOR signaling and remodeling. Mimics of microRNA-21-5p had opposing effects. IgE induced ASMC remodeling was significantly reduced by inhibition of mTOR or STAT3. In conclusion, non-immune IgE alone is sufficient for stimulated ASMC remodeling by upregulating microRNA-21-5p. Our findings suggest that the suppression of micoRNA-21-5p may present a therapeutic target to reduce airway wall remodeling.
31
Wiener, Edith, Roozina Rafique, Arnold Pizzey, Tomas Adejumo, George Chennell, and John B. Porter. "Upregulation of Hepatocellular NTBI Clearance Following Iron Loading Is Associated with Modulation of Membrane T- and L-Type Calcium Channels." Blood 118, no. 21 (November 18, 2011): 1038. http://dx.doi.org/10.1182/blood.v118.21.1038.1038.
Full textAbstract:
Abstract Abstract 1038 Background and Rationale. Under conditions of iron overload, the iron binding capacity of transferrin is exceeded and non-transferrin-bound iron (NTBI) species appear in the circulation, at concentrations typically within the range of 0.4–10 μM. NTBI species are rapidly cleared, mainly by the liver but also by the heart and endocrine tissues, causing iron overload of these organs. It is known that pre-loading of hepatocytes or cardiomyocytes with iron causes a paradoxical, dose and time-dependent, increase in NTBI clearance by these cells in vitro through mechanisms that are largely undetermined. In an iron overloaded mouse model, the uptake of ferrous iron by myocardium was related to the expression of L-type voltage–gated calcium channels and blockers of these channels reduced iron accumulation in the heart. However, it is unknown whether a similar mechanism underlies NTBI uptake by the liver. Previous work in our laboratory, using the human HuH7 hepatoma cell line, showed that increments in NTBI uptake that occurred following iron pre-loading of cells, were inhibited by the T-calcium channel inhibitor mibefradil. Methods. Flow cytometry and quantitative confocal microscopy were used to assess for changes in the abundance and subcellular distribution of L- and T-type calcium channels by iron-loading (ferric ammonium citrate [FAC]) of HuH7 hepatocytes. These cells were pre-incubated for 24 hours with increasing concentrations of FAC and then fixed (by 4% paraformaldehyde) or fixed and permeabilised (by 0.1% Triton X-100/PBS). Fixed HuH7 cells in suspension were immuno-stained with rabbit polyclonal antibodies against the alpha-1 subunits D (L-type) or H (T-type) and any changes in L- and T-type calcium channels on the cell surface measured by flow cytometry. Confocal microscopy was also performed on fixed and subsequently permeabilised adherent HuH7 cells, which were then immuno-stained by the same antibodies and subjected to fluorescence analysis using the Volocity program (Perkins Elmer) in order to assess intracellular distribution of these channels. Results. At clinically relevant pre-loading concentrations of 2 μM FAC, flow cytometry of fixed cells showed a 38% increase in T-type calcium channels on the outer membrane but no significant change in L-type channels. However, at higher FAC concentrations (20μM and 200 μM), there was a dose dependent rise in the membrane expression of L-type channels but a fall in T-type channels. With 200 μM FAC, the expression of the L-type channel was almost 3x that of the controls (p<0.0001) while the T-type channels were reduced to 65% of the untreated cells (p=0.03). Confocal microscopy of fixed and permeabilised cells showed similar effects on calcium channels following iron pre-loading. At. the clinically relevant concentrations of 2 μM iron, cellular images and line profiles exhibited greater over-all and outer membrane T-type fluorescence than controls, while L-type channels remained unaffected. By contrast, at higher pre-loading concentrations of FAC (20μM and 200μM), dose dependent increases in L-type fluorescence were observed, particularly on the cell membrane, whereas T-type fluorescence was less than that observed at 2 μM iron and similar to that of untreated cells. Conclusions. These findings show that previously demonstrated up-regulation of transferrin independent iron uptake into HuH7 hepatocytes, following cell pre-loading, are associated with an increase in membrane expression of T-type calcium channels at 2μM iron and of L-type channels at concentrations >20μM iron. Previous findings in our laboratory showing up-regulation of NTBI clearance under these conditions, but with no change in mRNA for L or T type channels, and with inhibition of this enhanced uptake by T-type channel blockers, suggest that these effects occur at a post-transcriptional level. These findings also indicate that use of selective calcium channel blockade could be useful in the treatment of iron overload conditions. Disclosures: No relevant conflicts of interest to declare.
32
Zini, Gina, Stefano Valentini, Pierluigi Puggioni, Tommaso Za, Antonella Di Mario, Carlo Rumi, and d'Onofrio Giuseppe. "Detection of the BCR-ABL Fusion protein2 by Using the Abbott Cell Dyn Sapphire.a Routine Blood Hematology Analyser." Blood 114, no. 22 (November 20, 2009): 4699. http://dx.doi.org/10.1182/blood.v114.22.4699.4699.
Full textAbstract:
Abstract Abstract 4699 Objectives We have evaluated the possibility to use the new BD Cytometric Bead Array to detect the presence of the BCR-ABL fusion protein2 in peripheral blood samples with a routine blood hematology analyser, the Abbott Cell-Dyn Sapphire, for a quick identification of those positive samples, both at diagnosis and at follow up to detect the MRD. This pilot study was carried out on 5 samples with different level of BCR-ABL fusion protein2 plus 2 control samples positive and negative respectively: results were compared with those obtained with the BD FACScanto and the standard FISH analysis reference method. Methods Flow cytometry allows for the discrimination of particles on the basis of attributes such as size and fluorescence. BD Cytometric Bead Array (CBA) systems provide a way of coupling a soluble analyte or set of analytes with beads of known size and fluorescence, making it possible to detect analytes through flow cytometry.1 The BD” BCR-ABL Protein Kit uses this technology to detect BCR-ABL fusion proteins2 in human blood research samples. Cell-Dyn Sapphire is a routine automated hematology analyzer for full blood count. Moreover the system employs immuno-fluorescence analysis technology, similar to that used on a dedicated fluorescence flow cytometer, for analysis of MAb applications. BD FacScant is a routine dedicated flow cytometer using 6 fluorescence channels. FISH (Fluorescence In Situ Hybridation) is a cytogenetic technique using fluorescent probes that bind to only those part of the chromosome with which they show a high degree of sequence similarity in interphase nuclei and on metaphase chromosome. FISH detects and localizes the presence, the absence or the translocation of specific DNA sequences on chromosomes. In this patients' category, LSI bcr/abl dual fusion DNA probe hybridises to chromosome 22q11.2 (breakpoint cluster region SpectrumGreen) and to chromosome 9q34 (abl oncogene SpectrumOrange). In interphase nuclei of normal cells, the probe signals generally appear as two distinct signals of each colour. In the pathological cells with bcr/abl translocation, individual orange and green signals from the normal 9 and 22 chromosome and two orange/green fusion signals (one each from the derivative 9 and 22 chromosomes) is observed. We have analysed 200 nuclei in fluorescence microscopy for each sample. Statistical analyses were performed using the MedCalc program comparing the median fluorescence values obtained by the 2 cytometers and with the standard diagnostic procedure. Results Correlation of median fluorescence data between the BD FACScanto and the CD Sapphire is excellent with a Correlation coefficient r = 1,0000 and a Significance level P<0,0001 (Fig 1). The Box –and –whisker Multiple comparison graph of the CD method versus the FISH reference one is represented in Figure 2. The results of our pilot study demonstrate the robustness of this new diagnostic tool in detecting the presence of the BCR-ABL fusion protein2 in the routine Laboratory of Hematology. The main limit to use this tool is now represented by the high cost of the BD Cytometric Bead Array. Disclosures: No relevant conflicts of interest to declare.
33
Reininger, Armin J., Harry F. G. Heijnen, Hannah Schumann, Wolfgang Schramm, and Zaverio M. Ruggeri. "Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress." Blood 106, no. 11 (November 16, 2005): 414. http://dx.doi.org/10.1182/blood.v106.11.414.414.
Full textAbstract:
Abstract We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis. Figure Figure
34
Haasemann, M., J. Cartaud, W. Muller-Esterl, and I. Dunia. "Agonist-induced redistribution of bradykinin B2 receptor in caveolae." Journal of Cell Science 111, no. 7 (April 1, 1998): 917–28. http://dx.doi.org/10.1242/jcs.111.7.917.
Full textAbstract:
Redistribution of receptors within the plasma membrane as well as between the plasma membrane and various cell compartments presents an important way of regulating the cellular responsiveness to their cognate agonists. We have applied immunocytochemical methods to localize the bradykinin B2 receptor and to examine its agonist induced redistribution in A431 cells. In situ labeling with antibodies to ectodomain-2 of the receptor which do not interfere with bradykinin binding of the receptor showed a random distribution of the B2 receptor on the plasma membrane. Stimulation of cells with 20 nM bradykinin markedly reduced the accessibility of the antibody to its corresponding epitope in non-permeabilized cells. Immuno-electron microscopy revealed the presence of receptors in membrane-near vesicles that are surrounded by an electron-transparent halo. Fluorescence microscopic double labeling co-localized the B2 receptor protein with caveolin-1 by a convergent pattern of punctate staining. At the ultrastructural level the B2 receptor protein was found in vesicles that bear the immunolabel of caveolin-1 and display the morphological characteristics of caveolae. We conclude that stimulation of B2 receptors results in their redistribution and sequestration in caveolae, an event that is likely to be implicated in receptor signaling and/or desensitization. The localization of B2 receptors in endosome-like structures after prolonged exposure to bradykinin might indicate that the internalization through caveolae may communicate with other endocytotic pathways of A431 cells.
35
Meijer, Alexander B., Sigrid D. Roosendaal, Vincent Limburg, Carmen van der Zwaan, Kees W. Rodenburg, and Koen Mertens. "LDL Receptor Mediates Effective Endocytosis of Activated Factor V." Blood 110, no. 11 (November 16, 2007): 1766. http://dx.doi.org/10.1182/blood.v110.11.1766.1766.
Full textAbstract:
Abstract The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.
36
Bochenek, Magdalena, Nico Rosinus, Mareike Lankeit, Lukas Hobohm, Felix Bremmer, Eva Schütz, Frederikus Klok, et al. "From thrombosis to fibrosis in chronic thromboembolic pulmonary hypertension." Thrombosis and Haemostasis 117, no. 04 (2017): 769–83. http://dx.doi.org/10.1160/th16-10-0790.
Full textAbstract:
SummaryThe pathomechanisms underlying the development of thrombofibrotic pulmonary artery occlusions in Chronic Thromboembolic Pulmonary Hypertension (CTEPH) are largely unknown. The aim of this study was to allocate distinct cellular processes playing a role in thrombus resolution, such as inflammation, hypoxia, proliferation, apoptosis and angiogenesis, to different stages of thrombofibrotic remodelling. A total of 182 pulmonary endarterectomy (PEA) specimens were collected from 31 CTEPH patients. To facilitate co-localisation, Tissue MicroArrays were prepared and processed for (immuno)-histochemistry and confocal fluorescence microscopy. Murine venous thrombus formation and resolution was examined after inferior vena cava ligation. PEA tissues exhibited five morphologically distinct regions predominantly consisting of either fibrin-, erythrocyte- or extracellular matrix-rich thrombus, myofibroblasts, vessels or fibrotic tissue, and were found to resemble chronological stages of thrombus resolution in mice. Cellularity was highest in vessel-rich regions, and numerous cells were strongly positive for HIF1α or HIF2α as well as markers of activated VEGF signalling, including endothelial nitric oxide synthase. On the other hand, negative regulators of angiogenic growth factor signalling and reactive oxygen species were also highly expressed. Immune cells, primarily macrophages of the M2 subtype and CD117 haematopoietic progenitors were detected and highest in vascularised regions. Our findings demonstrate the simultaneous presence of different stages of thrombus organisation and suggest that hypoxia-induced endothelial, mesenchymal and immune cell activation may contribute to thrombofibrosis in CTEPH. This systematic histological characterisation of the material obstructing pulmonary vessels in CTEPH may provide a valuable basis for further studies aimed at determining causal factors underlying this disease.Supplementary Material to this article is available online at www.thrombosis-online.com.
37
Schrödl, Kathrin, Hamza Oelmez, Martin Edelmann, Rudolf Maria Huber, and Albrecht Bergner. "Altered Ca2+-Homeostasis of Cisplatin-Treated and Low Level Resistant Non-Small-Cell and Small-Cell Lung Cancer Cells." Analytical Cellular Pathology 31, no. 4 (January 1, 2009): 301–15. http://dx.doi.org/10.1155/2009/495797.
Full textAbstract:
Background: Chemotherapy often leads to encouraging responses in lung cancer. But, in the course of the treatment, resistance to chemotherapy ultimately limits the life expectancy of the patient. We aimed at investigating if treatment with cisplatin alters the intracellular Ca2+-homeostasis of lung cancer cells and how this may be related to cisplatin resistance.Methods: The squamous cell lung carcinoma cell line EPLC M1 and the small cell lung cancer cell line H1339 were exposed to cisplatin analogue to the in vivo pharmacokinetics. Changes in the cytoplasmic Ca2+-concentration ([Ca2+]c) were recorded using fluorescence microscopy. Protein expression was quantified using immuno-fluorescence and Western Blot analysis. Changes in gene expression were accomplished by small-interfering (si) RNA techniques.Results: Four “cycles” of cisplatin led to low level resistance in EPLC and H1339 cells. In the low level resistant cell clones, the Ca2+-content of the endoplasmic reticulum (ER) was decreased. In low level resistant EPLC cells, this was correlated with an increased expression of the inositol-1,4,5-trisphosphate receptor (IP3R). Inhibiting the increased expression of IP3R using siRNA, the low level resistance could be reversed. In low level resistant H1339 cells, the decreased Ca2+-content of the ER was correlated with a decreased expression of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Decreasing the expression of SERCA in naïve H1339 cells resulted in low level cisplatin resistance.Conclusion: We conclude that cisplatin therapy leads to a decreased Ca2+-content of the ER thereby inducing low level resistance. This is caused by upregulation of the IP3R in EPLC and decreased expression of SERCA in H1339 cells.
38
Barwick, Shannon R., Mevish S. Siddiq, Jing Wang, Haiyan Xiao, Brendan Marshall, Elizabeth Perry, and Sylvia B. Smith. "Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells." Antioxidants 10, no. 6 (June 19, 2021): 981. http://dx.doi.org/10.3390/antiox10060981.
Full textAbstract:
Sigma 1 receptor (Sig1R), a modulator of cell survival, has emerged as a novel target for retinal degenerative disease. Studies have shown that activation of Sig1R, using the high affinity ligand (+)-pentazocine ((+)-PTZ), improves cone function in a severe retinopathy model. The rescue is accompanied by normalization of levels of NRF2, a key transcription factor that regulates the antioxidant response. The interaction of Sig1R with a number of proteins has been investigated; whether it interacts with NRF2, however, is not known. We used co-immunoprecipitation (co-IP), proximity ligation assay (PLA), and electron microscopy (EM) immunodetection methods to investigate this question in the 661W cone photoreceptor cell line. For co-IP experiments, immune complexes were precipitated by protein A/G agarose beads and immunodetected using anti-NRF2 antibody. For PLA, cells were incubated with anti-Sig1R polyclonal and anti-NRF2 monoclonal antibodies, then subsequently with (−)-mouse and (+)-rabbit PLA probes. For EM analysis, immuno-EM gold labeling was performed using nanogold-enhanced labeling with anti-NRF2 and anti-Sig1R antibodies, and data were confirmed using colloidal gold labeling. The co-IP experiment suggested that NRF2 was bound in a complex with Sig1R. The PLA assays detected abundant orange fluorescence in cones, indicating that Sig1R and NRF2 were within 40 nm of each other. EM immunodetection confirmed co-localization of Sig1R with NRF2 in cells and in mouse retinal tissue. This study is the first to report co-localization of Sig1R-NRF2 and supports earlier studies implicating modulation of NRF2 as a mechanism by which Sig1R mediates retinal neuroprotection.
39
Nykänen, Marko J., Marjatta Raudaskoski, Helena Nevalainen, and Anita Mikkonen. "Maturation of barley cysteine endopeptidase expressed inTrichoderma reeseiis distorted by incomplete processing." Canadian Journal of Microbiology 48, no. 2 (February 1, 2002): 138–50. http://dx.doi.org/10.1139/w01-144.
Full textAbstract:
Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley.Key words: cysteine proteinase, secretion, Kex2p, glycosylation, modified Golgi-like body.
40
Gadal, Olivier, Daniela Strauss, Elisabeth Petfalski, Pierre-Emmanuel Gleizes, Nicole Gas, David Tollervey, and Ed Hurt. "Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing." Journal of Cell Biology 157, no. 6 (June 10, 2002): 941–52. http://dx.doi.org/10.1083/jcb.200111039.
Full textAbstract:
Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.
41
Permann, Charlotte, Klaus Herburger, Martin Felhofer, Notburga Gierlinger, Louise A. Lewis, and Andreas Holzinger. "Induction of Conjugation and Zygospore Cell Wall Characteristics in the Alpine Spirogyra mirabilis (Zygnematophyceae, Charophyta): Advantage under Climate Change Scenarios?" Plants 10, no. 8 (August 23, 2021): 1740. http://dx.doi.org/10.3390/plants10081740.
Full textAbstract:
Extreme environments, such as alpine habitats at high elevation, are increasingly exposed to man-made climate change. Zygnematophyceae thriving in these regions possess a special means of sexual reproduction, termed conjugation, leading to the formation of resistant zygospores. A field sample of Spirogyra with numerous conjugating stages was isolated and characterized by molecular phylogeny. We successfully induced sexual reproduction under laboratory conditions by a transfer to artificial pond water and increasing the light intensity to 184 µmol photons m−2 s−1. This, however was only possible in early spring, suggesting that the isolated cultures had an internal rhythm. The reproductive morphology was characterized by light- and transmission electron microscopy, and the latter allowed the detection of distinctly oriented microfibrils in the exo- and endospore, and an electron-dense mesospore. Glycan microarray profiling showed that Spirogyra cell walls are rich in major pectic and hemicellulosic polysaccharides, and immuno-fluorescence allowed the detection of arabinogalactan proteins (AGPs) and xyloglucan in the zygospore cell walls. Confocal RAMAN spectroscopy detected complex aromatic compounds, similar in their spectral signature to that of Lycopodium spores. These data support the idea that sexual reproduction in Zygnematophyceae, the sister lineage to land plants, might have played an important role in the process of terrestrialization.
42
Symmons, M. F., S. R. Martin, and P. M. Bayley. "Dynamic properties of nucleated microtubules: GTP utilisation in the subcritical concentration regime." Journal of Cell Science 109, no. 11 (November 1, 1996): 2755–66. http://dx.doi.org/10.1242/jcs.109.11.2755.
Full textAbstract:
Microtubule assembly kinetics have been studied quantitatively under solution conditions supporting microtubule dynamic instability. Purified GTP-tubulin (Tu-GTP) and covalently cross-linked short microtubule seeds (EGS-seeds; Koshland et al. (1988) Nature 331, 499) were used with and without biotinylation. Under sub-critical concentration conditions ([Tu-GTP] < 5.3 microM), significant microtubule growth of limited length was observed on a proportion of the EGS-seeds by immuno-electron microscopy. A sensitive fluorescence assay for microtubule GDP production was developed for parallel assessment of GTP utilisation. This revealed a correlation between the detected microtubule growth and the production of tubulin-GDP, deriving from the shortening phase of the dynamic microtubules. This correlation was confirmed by the action of nocodazole, a specific inhibitor of microtubule assembly, that was found to abolish the GDP release. The variation of the GDP release with tubulin concentration (Jh(c) plot) was determined below the critical concentration (Cc). The GDP production observed was consistent with the elongation of the observed seeded microtubules with an apparent rate constant of 1.5 × 10(6) M-1 second-1 above a threshold of approximately 1 microM tubulin. The form of this Jh(c) plot for elongation below Cc is reproduced by the Lateral Cap model for microtubule dynamic instability adapted for seeded assembly. The behaviour of the system is contrasted with that previously studied in the absence of detectable microtubule elongation (Caplow and Shanks (1990) J. Biol. Chem. 265, 8935–8941). The approach provides a means of monitoring microtubule dynamics at concentrations inaccessible to optical microscopy, and shows that essentially the same dynamic mechanisms apply at all concentrations. Numerical simulation of the subcritical concentration regime shows dynamic growth features applicable to the initiation of microtubule growth in vivo.
43
Wang, Jishi, Xiaoyan Hu, Xinmei Liu, Yuan Yang, Yingya Wu, and Qin Fang. "The Anticancer Effect of Cytochrome P450 2E1 Gene Which Was Mediated by Recombination Adenovirus in Mesenchymal Stem Cells." Blood 108, no. 11 (November 16, 2006): 4737. http://dx.doi.org/10.1182/blood.v108.11.4737.4737.
Full textAbstract:
Abstract Dacarbazine (DTIC) can inhibit purine nucleotide synthesis after activated by P4502E1(CYP2E1) in liver and can be used for the treatment of malignant melanoma and Hodgkins lymphoma. Objective: To explore the anticancer effect of the combination of CYP2E1 and DTIC. Methods: A full length cDNA fragment encoding the human CYP2E1 was cloned and was transfected into bone marrow mesenchymal stem cells (BMSCs) through recombinant adenovirus vector. The recombinant adenovirus vectors pAd5CMV-CYP2E1 and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method. The expression of CYP2E1 gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP. In vitro, gene transfected BMSCs were co-cultured with DTIC, and then the supernatant was added into the medium of human malignant melanoma M14 cells. Fluorescence microscopy, DNA Ladder, flowcytometry, electrophoresis were employed to tests the apoptosis, and MTT method was applied to detecting the IC50. In the animal experiment, the CYP2E1 gene tranfected BMSCs were injected in combination with DTIC directly into the malignant melanomas on nude mice or through caudal vein, and the injected BMSCs transfected pAd5CMV-EGFP and DTIC in the same manner into nude control mice. The inhibiting effect of new planted tumors of experimental group was monitored through Immuno-histochemistry analysis and the shape and size of tumors were measured. CYP2E1-EGFP gene tranfected BMSCs were injected into nude mice through caudal vein and tracked by fluorescence. Results: CYP2E1 gene(1482bp) was cloned successfully and it was conformed by DNA sequencing; Endonuclease and PCR analyses showed that recombinant adenovirus vector had been constructed and high titer recombinant adenovirosome(1×1012pfu/ml) had been packaged successfully. BMSCs had been isolated and flowcytometry showed their phenotype were CD44(+),CD105(+), CD34(−)and CD45(−). The mRNA and protein quantities of the group of CYP2E1 gene tranfected BMSCs were significantly higher than the control group (P<0.05, n=24). In vitro, immunity fluorescence microscope, DNA Ladder, and flowcytometry all showed the supernatant of co-culture of CYP2E1 gene tranfected BMSCs and DTIC) significantly decreased the mortality of M14 cells compared with pAd5CMV-EGFP-BMSCs control group (P < 0. 05, n=24). In vivo, the sizes of the tumor in the nude mice treated with transfected BMSCs and DTIC were significantly smaller than control case and changed along with concentration. (P< 0.05, n=20). No new tumor was found in the nude mouse inoculated with M14 cells after the injection of CYP2E1 transfected BMSCs and DTIC. Conclusion: Recombination adenovirus vector was able to express CYP2E1 in BMSCs. Gene CYP2E1 transfected BMSCs and DTIC showed anticancer activity both in vitro and vivo.
44
Wierenga, Pieter K., Isabelle Lombaert, Willy Visser, Harm H. Kampinga, Gerald de Haan, and Rob P. Coppes. "Bone Marrow-Derived Stem Cells Reduce Radiation-Induced Damage to Salivary Glands." Blood 104, no. 11 (November 16, 2004): 1194. http://dx.doi.org/10.1182/blood.v104.11.1194.1194.
Full textAbstract:
Abstract The salivary glands are often included in the field of irradiation during radiotherapy of head and neck cancer. This can result in severe side-effects that reduces the quality of life of the patient and may even limit the treatment dose. Late damage to the salivary glands is mainly caused by exhaustion of the tissue specific stem cells. Post-irradiation replacement of salivary gland stem cells with donor stem cells may ameliorate radiation-induced complications. Bone marrow-derived stem cells (BMSC) have been shown to be multipotent and thereby able to engraft in many tissues after injury. In this study, we assessed the potential of BMSC to reduce irradiation-induced salivary gland damage. C57BL/6 mice were transplanted with bone marrow from eGFP transgenic animals. After two months the salivary glands of these chimeric mice were locally irradiated with 15 Gy. BMSC were mobilized 10, 30 and/or 60 days after irradiation by s.c. injection of rHu-PEG-G-CSF. Saliva secretion (μl/15 minutes) was measured up to 90 days after irradiation by pilocarpine induction. Hereafter, the glands were extirpated and examined for eGFP-expression. From every individual animal one parotid and one submandibular gland was used to prepare single cell suspensions in order to detect eGFP-positve cells by flow cytometry. The other parotid and submandibular glands were analyzed using confocal laser fluorescence scanning microscopy and light microscopy. G-CSF treatment yielded in an increase of saliva flow for all time points. The optimal time-point for mobilization, however, was 30 days after irradiation as is demonstrated by an improvement of salivary flow from 5 to 30% when compared to radiation alone. FACS analysis showed that up to 10% of the isolated cells were eGFP-positive. Microscopic analysis revealed a similar amount of positive cells and an improved morphology. Immuno-histochemistry using anti-SM-actin antibodies showed the close vicinity of actin and eGFP within the cells, demonstrating the occurence of BMSC derived myoepithelial cells in irradiated salivary glands. Furthermore, using cell-type specific antibodies, the meyoepethilial nature of the eGFP positive was revealed. In conclusion, the results show that BMSC home to severely damaged salivary glands after mobilization. Hence, BMSC mobilization could become a promising modality to ameliorate radiation-induced complications in salivary glands after radiotherapy.
45
Paredes-Santos, T. C., E. S. Martins-Duarte, W. de Souza, M. Attias, and R. C. Vommaro. "Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro." Parasitology 145, no. 8 (November 28, 2017): 1027–38. http://dx.doi.org/10.1017/s0031182017002050.
Full textAbstract:
AbstractToxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a ‘cage’ around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.
46
Brouckova, Adela, and Karel Holada. "Cellular prion protein in blood platelets associates with both lipid rafts and the cytoskeleton." Thrombosis and Haemostasis 102, no. 11 (2009): 966–74. http://dx.doi.org/10.1160/th09-02-0074.
Full textAbstract:
SummaryThe recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.
47
Barkovskaya, M. Sh, A. G. Bogomolov, N. Yu Knauer, N. B. Rubtsov, and V. A. Kozlov. "Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology." Journal of Bioinformatics and Computational Biology 15, no. 02 (April 2017): 1650041. http://dx.doi.org/10.1142/s0219720016500414.
Full textAbstract:
Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (C3TA[Formula: see text] peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated.
48
Yin, Wei, Chun Wang, Kuohai Fan, Na Sun, Yaogui Sun, and Hongquan Li. "In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages." PeerJ 7 (March 8, 2019): e6439. http://dx.doi.org/10.7717/peerj.6439.
Full textAbstract:
Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced.
49
Van Blerkom, Jonathan, Patrick Davis, John Merriam, and Jane Sinclair. "Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human." Human Reproduction Update 1, no. 5 (January 1, 1995): 429–61. http://dx.doi.org/10.1093/humupd/1.5.429.
Full textAbstract:
Abstract This report describes spatial and temporal aspects of sperm penetration and intracytoplasmic migration, pronuclear evolution and the specificity of presyngamic opposition, stage-specific changes in cytoskeletal organization and the relative contribution of maternal and paternal components to mitotic spindle formation. These studies involved observations of living human oocytes during conventional insemination in vitro and after intracytoplasmic deposition of spermatozoa, analysis of chromatin organization and distribution during pronuclear evolution, and detection of actin and α-, rβ- and γ-tubulin by confocal immuno-fluorescence microscopy. Immature and mature oocytes, penetrated but unfertilized oocytes, fertilized but arrested eggs, and cleavage-stage embryos from normal and dispermic fertilizations were examined. The results demonstrate that sperm nuclear migration to the maternal perinuclear region is rapid and linear, occurs in the absence of a detectable cytoskeletal system and appears to be assisted by an unusual configuration of the sperm tail principal piece which results from either retained intracytoplasmic motility or the process by which the sperm tail is progressively incorporated into the oocyte. Our findings also show a specificity of pronuclear alignment that is associated with a polarized distribution of both maternal and paternal chromatin, and with the position of the sperm centrosome and the presence of microtubules nucleated from this structure. The results also indicate that a maternal microtubule nucleating capacity is present in the immature oocyte but is apparently inactive until spindle formation. The poles of the first mitotic spindle appear to be derived from the sperm centrosome, although some maternal contribution cannot be excluded. The sperm tail and centrosome persist in a single cell through the cleavage stages, and the latter serves as a prominent site of cytoplasmic microtubule nucleation. The results provide a detailed understanding of the cellular and nuclear morphodynamics of the human fertilization process and indicate subtle defects that may be responsible for early developmental failure.
50
Klaus, S., M. Ely, D. Encke, and G. Heldmaier. "Functional assessment of white and brown adipocyte development and energy metabolism in cell culture. Dissociation of terminal differentiation and thermogenesis in brown adipocytes." Journal of Cell Science 108, no. 10 (October 1, 1995): 3171–80. http://dx.doi.org/10.1242/jcs.108.10.3171.
Full textAbstract:
We investigated the effect of insulin, triiodothyronine (T3) and dexamethasone (a synthetic glucocorticoid) on differentiation, lipid metabolism and thermogenesis of preadipocytes isolated from white fat (WAT) and brown fat (BAT) from the Siberian dwarf hamster (Phodopus sungorus). Cell cultures from WAT and BAT were chronically treated with the above hormones alone or in any combination. After differentiation (day 8 or 9 of culture) we measured the following parameters: adipogenic index (number × size of adipocytes), protein content, lipolysis, cell respiration, and expression of the uncoupling protein UCP, which is unique to mitochondria of brown adipocytes. Insulin was the most important adipogenic factor for brown and white adipocytes and necessary for terminal differentiation, whereas dexamethasone alone completely inhibited differentiation. T3 had no effect on adipogenesis in WAT cultures, but further increased insulin stimulated adipogenesis in BAT cultures. Basal lipolysis was higher in WAT than in BAT cultures except when dexamethasone was present, which stimulated lipolysis in both culture types to the same extent. T3 had a pronounced dose dependent lipolytic effect on WAT cultures but very little effect on BAT cultures. Respiration rates were generally higher in differentiated adipocytes than in fibroblast like cells. T3 had no effect on thermogenesis in WAT cultures but increased thermogenesis in BAT cultures, and this was further elevated by insulin. UCP expression in BAT cultures could be detected by western blot in insulin treated, T3 treated and insulin+T3 treated cultures with highest expression in the latter. These results imply a possible dissociation of terminal differentiation and thermogenic function of brown adipocytes. In WAT cultures there was also a low level of UCP detectable in the insulin+T3 treated cultures. Immuno-fluorescence microscopy analysis revealed the presence of UCP in 10–15% of adipocytes from WAT cultures (in BAT cultures: 90%), indicating the presence of some brown preadipocytes in typical WAT deposits.