Permeabilization of microorganisms by Triton X-100

Miozzari, Giuseppe; Niederberger, Peter; ̈tter, R. Hu

doi:10.1016/0003-2697(78)90026-x

Cited by 191 publications

(85 citation statements)

References 26 publications

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“…peptides, siRNA, ODNs, proteins), and results of this assay can be predictive of performance as an intracellular drug delivery vehicle. 3,4,8,18,19 Thus, this assay represents an effective screen to gauge the ability of polymeric drug carriers to mediate intracellular drug delivery based on their pH-dependent membrane disruption.…”

Section: Discussionmentioning

confidence: 99%

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

JoVE

274

245

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Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems.In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

JoVE

274

245

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“…As particularly small proteins (< 80 kDa, e.g. Tps1) may be lost during sample preparation in permeabilized cells (Miozzari et al, 1978), use of cell extracts was clearly the method of choice in the present study, where the occurrence of free Tps1 in the extracts of some mutant strains could be anticipated. From these observations it is obvious that the Tre6P synthase activities measured in vitro often do not reflect the actual in vivo situation.…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Reinders

Bürckert

Hohmann

et al. 1997

Molecular Microbiology

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SummarySynthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/ phosphatase complex in exponentially growing, heatshocked cells.

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“…The activity of neutral trehalase was measured in permeabilized cells (Miozzari et al, 1978). 40-ml aliquots of logphase cultures were filtered (Whatman GF/C), suspended in 1 ml 0.2 M Tricine (Na'), pH 7.0, 0.05% Triton X-100 and immediately frozen in liquid nitrogen.…”

Section: Enzyme Assays and Determination Of Metabolite Levelsmentioning

confidence: 99%

The role of trehalose synthesis for the acquisition of thermotolerance in yeast

Virgilio

Hottiger

Domínguez

et al. 1994

European Journal of Biochemistry

303

201

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In the yeast Saccharomyces cerevisiae, accumulation of the non-reducing disaccharide trehalose is triggered by various stimuli that activate the heat-schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat-induced and stationary-phase-induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPSl or TPS2, encoding subunits of the trehalose-6-phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat-shock or upon initiation of the stationary phase and significantly reduced the levels of heat-induced and stationary-phase-induced thermotolerance. Deletion of NTHZ, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat-induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T

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Permeabilization of microorganisms by Triton X-100

Cited by 191 publications

References 26 publications

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

The role of trehalose synthesis for the acquisition of thermotolerance in yeast

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