scholarly journals Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells.

1979 ◽  
Vol 83 (2) ◽  
pp. 511-515 ◽  
Author(s):  
N M Hogg ◽  
B Rotman
Keyword(s):  
Cell Surface ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Covalent Attachment ◽  
Histological Evidence ◽  
Coupling Procedure ◽  
Specific Affinity ◽  
Enzyme Label ◽  
Membrane Components

An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.

Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

2018 ◽  
Vol 475 (10) ◽  
pp. 1773-1788 ◽  
Author(s):  
Ditlev Birch ◽  
Malene V. Christensen ◽  
Dan Staerk ◽  
Henrik Franzyk ◽  
Hanne Mørck Nielsen
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Proliferating Cells ◽  
Epithelial Integrity ◽  
Cell Penetrating ◽  
Culture Models ◽  
Well Differentiated

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18–20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

scholarly journals The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1974 ◽  
Vol 140 (3) ◽  
pp. 405-411 ◽  
Author(s):  
Charles A. Pasternak ◽  
Kingsley J. Micklem
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Sendai Virus ◽  
Membrane Integrity ◽  
Intracellular Metabolites ◽  
Nitrophenyl Phosphate ◽  
Biochemical Studies ◽  
Membrane Components ◽  
Interesting System

1. Phospholipids prelabelled with [14C]acetate, [32P]phosphate, [3H]- or [14C]-choline or [3H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [3H]fucose, [14C]galactose or [3H]glucosamine. Less than 1nmol of lysophosphatidylcholine/107 cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K+ leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca2+ into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G1 and S periods of the cell cycle. 5. Leakage is inhibited by Ca2+, but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

scholarly journals Biological Effects of XyloCore, a Glucose Sparing PD Solution, on Mesothelial Cells: Focus on Mesothelial-Mesenchymal Transition, Inflammation and Angiogenesis

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2282
Author(s):  
Valentina Masola ◽  
Mario Bonomini ◽  
Maurizio Onisto ◽  
Pietro Manuel Ferraro ◽  
Arduino Arduini ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Viability ◽  
Biological Effects ◽  
Membrane Integrity ◽  
Glucose Content ◽  
Mesenchymal Transition ◽  
Low Glucose ◽  
Endothelial To Mesenchymal Transition ◽  
Osmotic Agents

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.

scholarly journals The Effects of CAMPATH-1H on Cell Viability Do Not Correlate to the CD52 Density on the Cell Surface

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e103254 ◽  
Author(s):  
Fuiyee Lee ◽  
Martha Luevano ◽  
Paul Veys ◽  
Kwee Yong ◽  
Alejandro Madrigal ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Viability

scholarly journals Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor:  Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

2007 ◽  
Vol 129 (2) ◽  
pp. 268-269 ◽  
Author(s):  
Siwarutt Boonyarattanakalin ◽  
Jianfang Hu ◽  
Sheryl A. Dykstra-Rummel ◽  
Avery August ◽  
Blake R. Peterson
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cell Surface Receptor ◽  
Synthetic Cell ◽  
Tissue Targeting ◽  
Surface Receptor

Cell surface interactions in conjugation: Tetrahymena ciliary membrane vesicles

1984 ◽  
Vol 4 (4) ◽  
pp. 681-687
Author(s):  
B Love ◽  
M B Rotheim
Keyword(s):  
Cell Surface ◽  
Mating Type ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Surface Interactions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ciliary Membrane ◽  
Adhesion Sites ◽  
Cell Surface Interactions

Tetrahymena ciliary membrane vesicles are shown to interact with preconjugant cells in a mating type-specific way. When cells are treated with vesicles of a different mating type before mixing for conjugation, cell pairing is enhanced, and the normal prepairing period is partially eliminated. This enhancement is mating type specific since it is not observed after pretreatment of cells with vesicles of their own mating type. In contrast, when vesicles are added at the time of mixing of two starved cultures, cell pairing is delayed in a concentration-dependent manner. By varying the conditions, we demonstrated enhancement or inhibition, or both. These results are interpreted in terms of two independent interactions of cells with vesicles. We suggest that first, vesicles substitute for another cell in cell-cell prepairing interaction and second, vesicles compete for adhesion sites produced during the prepairing period. Finally, the data presented are summarized within a speculative framework that calls attention to potential analogies with hormone-receptor signaling in mammalian cells.

scholarly journals An Alternative Coupling Procedure for Preparing Activated Sepharose for Affinity Chromatography of Penicillinase

1976 ◽  
Vol 29 (4) ◽  
pp. 305 ◽  
Author(s):  
RG Coombe ◽  
AM George
Keyword(s):  
Affinity Chromatography ◽  
Room Temperature ◽  
Cyanogen Bromide ◽  
Dry Weight ◽  
Enzymic Activity ◽  
Covalent Attachment ◽  
Affinity Column ◽  
Coupling Procedure ◽  
Coupling Techniques ◽  
Cyanogen Bromide Activation

Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axtm et al. (1967). Porath and Sundberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. a-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8� 0-8� 6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41 % of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.

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