Redifferentiation, cellular elongation and the cell surface during lens regeneration

Development ◽  
1977 ◽  
Vol 39 (1) ◽  
pp. 23-43
Author(s):  
Sharon Hornsby ◽  
Sara E. Zalik
Keyword(s):  
Cell Surface ◽  
Cytochalasin B ◽  
Fiber Formation ◽  
Lens Fiber ◽  
Neuraminidase Treatment ◽  
Lens Regeneration ◽  
Specific Proteins ◽  
Ionic Double Layer ◽  
Vinblastine Sulfate

(1) Dorsal irises from normal and lentectomized eyes of the newt Notophthalmus viridescens were cultured in vitro in the presence of colchicine (1·25 × 10−3 to 2·5 × 10−6 M), vinblastine sulfate (4·4 × 10−8) and cytochalasin B (20µg/ml). Explants were treated for 24 h and implanted into host eyes of previously lentectomized newts. (2) In regenerates treated with colchicine at stages prior to the onset of lens fiber formation, cellular elongation was inhibited or considerably delayed. Cytochalasin B has no effect on lens fiber differentiation when compared with regenerates treated with its solvent, dimethyl sulfoxide. (3) Colchicine-treated regenerates showed no detectable fluorescence when stained with anti-newt lens fluorescent antibodies, suggesting that cells in which cellular elongation was inhibited were unable to synthesize lens specific proteins. (4) Ultrastructural observations of elongating lens regenerate cells showed microtubules present in the cytoplasm parallel to the axis of elongation. Microtubules were not observed in depigmented cells from colchicine-treated regenerates. Characteristic vinblastine-induced microtubule crystals were present in depigmented cells from vinblastine-treated regenerates. (5) Experiments using cell electrophoresis showed that neuraminidase-sensitive groups were detectable at the ionic double layer of cells from implanted regenerates at stages where lens fiber differentiation occurs. Cells from colchicine-treated regenerates showed no significant reduction of their electrophoretic mobility after neuraminidase treatment. This suggests that the appearance of some cell surface components in the redifferentiated cell may also be colchicine sensitive and probably mediated by microtubules.

scholarly journals In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle

1999 ◽  
Vol 342 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Jeffrey W. RYDER ◽  
Yuichi KAWANO ◽  
Alexander V. CHIBALIN ◽  
Jorge RINCÓN ◽  
Tsu-Shuen TSAO ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transport ◽  
Transport System ◽  
Soleus Muscle ◽  
Cytochalasin B ◽  
Transport Activity ◽  
Wild Type ◽  
Glucose Transport System ◽  
Vitro Analysis

We have characterized the glucose-transport system in soleus muscle from female GLUT4-null mice to determine whether GLUT1, 3 or 5 account for insulin-stimulated glucose-transport activity. Insulin increased 2-deoxyglucose uptake 2.8- and 2.1-fold in soleus muscle from wild-type and GLUT4-null mice, respectively. Cytochalasin B, an inhibitor of GLUT1- and GLUT4-mediated glucose transport, inhibited insulin-stimulated 2-deoxyglucose uptake by > 95% in wild-type and GLUT4-null soleus muscle. Addition of 35 mM fructose to the incubation media was without effect on insulin-stimulated 3-O-methylglucose transport activity in soleus muscle from either genotype, whereas 35 mM glucose inhibited insulin-stimulated (20 nM) 3-O-methylglucose transport by 65% in wild-type and 99% in GLUT4-null mice. We utilized the 2-N-4-1-(1-azi-2,2,2-t r i f l u o r o e t h y l ) b e n z o y l - 1, 3 - b i s (D - m a n n o s e - 4 - y l o x y ) - 2 - p ro p y lamine (ATB-BMPA) exofacial photolabel to determine if increased cell-surface GLUT1 or GLUT4 content accounted for insulin-stimulated glucose transport in GLUT4-null muscle. In wild-type soleus muscle, cell-surface GLUT4 content was increased by 2.8-fold under insulin-stimulated conditions and this increase corresponded to the increase in 2-deoxyglucose uptake. No detectable cell-surface GLUT4 was observed in soleus muscle from female GLUT4-null mice under either basal or insulin-stimulated conditions. Basal cell-surface GLUT1 content was similar between wild-type and GLUT4-null mice, with no further increase noted in either genotype with insulin exposure. Neither GLUT3 nor GLUT5 appeared to account for insulin-stimulated glucose-transport activity in wild-type or GLUT4-null muscle. In conclusion, insulin-stimulated glucose-transport activity in female GLUT4-null soleus muscle is mediated by a facilitative transport process that is glucose- and cytochalasin B-inhibitable, but which is not labelled strongly by ATB-BMPA.

scholarly journals Adeno-associated virus mediated gene delivery: Implications for scalable in vitro and in vivo cardiac optogenetic models

2017 ◽  
Author(s):  
Christina M. Ambrosi ◽  
Gouri Sadananda ◽  
Aleksandra Klimas ◽  
Emilia Entcheva
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Transgene Expression ◽  
Neonatal Rat ◽  
Cell Surface Receptor ◽  
Specific Gene ◽  
Neuraminidase Treatment ◽  
Rat Hearts

ABSTRACTAimsAdeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in cardiac models relevant to optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models for cardiac optogenetics and elucidate the mechanism of virus uptake.MethodsIn vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry.ResultsTransgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV 1/6 (cell surface sialic acid) and AAV9(37/67kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with neuraminidase treatment yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the adult and neonatal rat hearts, but no expression in vitro in cultured cells. The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue slices.ConclusionThe combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts.

scholarly journals The endothelial glycocalyx anchors von Willebrand factor fibers to the vascular endothelium

2018 ◽  
Vol 2 (18) ◽  
pp. 2347-2357 ◽  
Author(s):  
Thejaswi Kalagara ◽  
Tracy Moutsis ◽  
Yi Yang ◽  
Karin I. Pappelbaum ◽  
Anne Farken ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Dynamic Change ◽  
Fiber Formation ◽  
Endothelial Glycocalyx ◽  
Endothelial Cell Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανβ3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber–dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

Elucidating the anti-biofilm and anti-quorum sensing potential of selenocystine against respiratory tract infections causing bacteria: in vitro and in silico studies

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Bharti Patel ◽  
Subrata Mishra ◽  
Indira K. Priyadarsini ◽  
Sirisha L. Vavilala
Keyword(s):  
Quorum Sensing ◽  
Respiratory Tract ◽  
Protein Function ◽  
Docking Studies ◽  
Klebsiella Pneumonia ◽  
Potential Candidate ◽  
In Silico Studies ◽  
Specific Proteins ◽  
Tract Infections

Abstract Bacteria are increasingly relying on biofilms to develop resistance to antibiotics thereby resulting in their failure in treating many infections. In spite of continuous research on many synthetic and natural compounds, ideal anti-biofilm molecule is still not found thereby warranting search for new class of molecules. The current study focuses on exploring anti-biofilm potential of selenocystine against respiratory tract infection (RTI)-causing bacteria. Anti-bacterial and anti-biofilm assays demonstrated that selenocystine inhibits the growth of bacteria in their planktonic state, and formation of biofilms while eradicating preformed-biofilm effectively. Selenocystine at a MIC50 as low as 42 and 28 μg/mL effectively inhibited the growth of Klebsiella pneumonia and Pseudomonas aeruginosa. The antibacterial effect is further reconfirmed by agar cup diffusion assay and growth-kill assay. Selenocystine showed 30–60% inhibition of biofilm formation in K. pneumonia, and 44–70% in P. aeruginosa respectively. It also distorted the preformed-biofilms by degrading the eDNA component of the Extracellular Polymeric Substance matrix. Molecular docking studies of selenocystine with quorum sensing specific proteins clearly showed that through the carboxylic acid moiety it interacts and inhibits the protein function, thereby confirming its anti-biofilm potential. With further validation selenocystine can be explored as a potential candidate for the treatment of RTIs.

scholarly journals Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1929
Author(s):  
Tereza Cervena ◽  
Andrea Rossnerova ◽  
Tana Zavodna ◽  
Jitka Sikorova ◽  
Kristyna Vrbova ◽  
...  
Keyword(s):  
Cytochalasin B ◽  
Culture Media ◽  
Methodological Approach ◽  
Micronucleus Assay ◽  
Toxicity Testing ◽  
In Vitro Toxicity ◽  
Testing Strategies ◽  
Transmission Electron ◽  
The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

scholarly journals Cell Surface Expression of Nrg1 Protein in Candida auris

2021 ◽  
Vol 7 (4) ◽  
pp. 262
Author(s):  
Anuja Paudyal ◽  
Govindsamy Vediyappan
Keyword(s):  
Cell Surface ◽  
Growth Conditions ◽  
Zinc Ion ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Unexpected Finding ◽  
Candida Auris ◽  
Cell Surface Proteins ◽  
Biofilm Growth

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

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