Topic
Lysis
About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.
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TL;DR: The critical phenomenon determining antifungal effect of cationic surfactants and lipids is not cell lysis but rather the change of cell surface charge from negative to positive.
Abstract: Objectives: To determine the mechanism of antimicrobial action for cationic lipid dioctadecyldimethylammonium bromide (DODAB) and hexadecyltrimethylammonium bromide (CTAB) against Candida albicans. Methods: Determination of DODAB or CTAB adsorption isotherms; cell viability; cell electrophoretic mobility (EM); and leakage of small phosphorylated compounds, proteins or DNA from fungus or haemoglobin from erythrocytes. Results: High affinity isotherms for CTAB and DODAB adsorption onto fungus cells (10 8 cfu/mL) yield limiting adsorption at 7.8 and 3.7 · 10 9 molecules per cell, respectively. Negatively charged C. albicans cells (10 6 cfu/mL) remain viable whereas positively charged ones die. At 0.3 mM CTAB or 0.01 mM DODAB, EM is zero and fungus viability is 50%. Cells start to die at submicellar CTAB concentrations and fungus lysis does not play a significant role in the mechanism of antifungal action. Over 0.1–10 mM CTAB or DODAB, there is no leakage of tested compounds from C. albicans cells despite the low cell viability. In contrast to the fungus, under isotonic conditions, cationic amphiphiles induce haemolysis over a range of low DODAB (>0.01 mM) and CTAB (>0.001 mM) concentrations. Conclusions: The critical phenomenon determining antifungal effect of cationic surfactants and lipids is not cell lysis but rather the change of cell surface charge from negative to positive.
197 citations
TL;DR: Time-resolved imaging was used to examine the use of pulsed laser microbeam irradiation to produce cell lysis and indicated that cells subject to transient shear stresses in excess of a critical value were lysed while cells exposed to lowerShear stresses remained adherent and viable.
197 citations
TL;DR: A protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts is described.
Abstract: The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.
197 citations
TL;DR: It is found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels in real-time qRT-PCR.
194 citations
TL;DR: It is reported here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores, and is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems.
Abstract: Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1–secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1–dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1–mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1–mediated lysis.
194 citations