Permeabilization

Efficient downstream processing of products is facilitated when the product can be recovered from the medium. Besides by elicitation and immobilization, excretion of the product can be obtained by permeabilization of the cells. Various strategies have been used chemical permeabilization, electrical permeabilization, ultrasonic permeabilization, and ion-tophoretic permeabilization 111). Chemical permeabilization comprises the use of, among others, organic solvents as DMSO and chloroform and surface active chemicals (as Triton X-100). Results with the various tech- 

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Nisin acts bactericidally primarily against gram-positive bacteria. It acts best at acid pH and is almost insoluble at physiological pH. Nisin and probably all lantibiotics appear to permeabilize the bacterial ceU membrane to release small molecules, resulting in an immediate coUapse of the membrane... 

Procedures of the beta-galactosidase activity measuring using colour reaction with ONPG and X-Gal without cells permeabilization were developed and the detection limit at the level of 4 ppb has been achieved. The influence of the foreign ions (phosphate, sulphate, carbonate et. al) was studied. 

Although in in vivo circumstances an intracellular free calcium increase apparently acts as the primary modulator of contraction, it can be bypassed in highly permeabilized smooth muscle preparations where the active subunit of MLCK can be introduced to phosphorylate myosin and induce contraction. The MLCK catalyzed phosphorylation of serine-19 is seen as the necessary event in the activation of smooth muscle myosin to form crossbridges. Thus, the rising phase of force during an isometric smooth muscle contraction follows an increase in the degree of phosphorylation of myosin, and that in turn follows the transient rise of (a) cytosolic free Ca, (b) Ca-calmodulin complexes, and (c) the active form of MLCK. The regulation of the intracellular calcium is discussed below. The dynam-... 

Figure 3. The binding and dissociation of FU EP and receptor on permeabilized neutrophils at 37 C The data are plotted as the specific binding (pmoles FLPEP bound/lO cells) on a log plot versus time. 10 permeabilized cells/mL were exposed to 1 nM FLPEP at time 0. After IS or 120 s, antibody to fluorescein was added to each sample. 60 seconds later, 10- M GTP yS was added. (Reproduced with permission from reference 22. Copyright 1987 Journal of Biological Chemistry.)...

Figure 4 shows an analysis of binding and dissociation of FLPEP in permeabilized cells in the absence (top panel) and the presence of GTPyS (lower panel). Dissociation is initiated, as indicated, by the addition of a receptor antagonist, tBoc-phe-leu-phe-leu-phe. The solid line is a fit to the data. The data from this experiment have... 

Figure 4. Continuous analysis by fluorescence polarization of FLPEP binding and dissociation to permeabilized neutrophils. Each curve represents the average of two determinations. The descending curves represent the effect of the addition of receptor antagonist after FLPEP had bound to the receptors. Upper panel, in the absence of GTPyS, receptor antagonist added at t = 60 s lower panel, in the presence of GTPyS, antagonist added at t =...

Adtveoitages. The antibody technology is remarkably convenient. It requires only that a significant fraction of FLPEP be receptor bound ( 10%). The assay can be set up in a few minutes and is applicable to membranes, permeabilized cells, cells, or any other preparation in which the receptor concentration is in the range of 1 niV or greater. We use this assay routinely to analyze ligand dissociation kinetics. 

Cell permeabilization and uptake of anti-sense peptide-peptide nucleic acid (PNA) into Escherichia coli. J. Biol. Chem. 2002 277 7144-7147. 

In the interdisciplinary field of biophysics and biotechnology, the bioeffects of electric field have received considerable interest for both fundamental studies on these interaction mechanisms and potential application. However, the effects of pulsed electric field (PEF) on secondary metabolism in plant cell cultures and fermentation processes have been unknown. Therefore, it would be very interesting to find out whether PEF could be used as a new tool for stimulating secondary metabolism in plant cell cultures for potential application to the value-added plant-specific secondary metabolite production. Furthermore, if the PEF permeabilization and elicitation are discovered in a cell culture system, the combination of... 

Chelators, especially ethylenediamine tetra-acetic acid (EDTA), have been used as potentiators of the aetion of ehloroxylenol. Vaara has extensively reviewed the subject of permeabilization and Ayres, Furr and Russell have described a rapid method of evaluating the permeabilizahon of Ps. aeruginosa (see Further Reading, Sechon 6). 

Ayres H., Furr J.R. Russell A.D. (t993) A rapid method of evaluating permeabilizing activity against Pseudomonas aeruginosa. Lett Appl Microbiol, 11, i49-t87. 

The A20 antibody did not bind significantly to native SR vesicles, but solubilization of the membrane with C Eg or permeabilization of the vesicles by EGTA exposed its epitope and increased the binding more than 20-fold [139], By contrast, the A52 antibody reacted freely with the native sarcoplasmic reticulum, while the A25 antibody did not react either in the native or in the C Eg solubilized or permeabilized preparations, and required denaturation of Ca " -ATPase for reaction, Clarke et al, [139] concluded that the epitope for A52 is freely exposed on the cytoplasmic surface, while the epitope for A20 was assigned to the luminal surface, where it became accessible to cytoplasmic antibodies only after solubilization or permeabilization of the membrane. The epitope for A25 is assumed to be on the cytoplasmic surface in a folded structure and becomes accessible only after denaturation. 

The kinetics of fructose phosphorylation catalyzed by permeabilized membranes [48],... 

The two substrate kinetics of the overall reaction catalyzed by the complex in permeabilized membranes showed classical ping-pong kinetics in accordance with a phosphorylated enzyme intermediate. The affinity constants for fructose and P-enolpyruvate were 8 and 25 /iM, respectively. 

Toxicity or inhibition of biodegradation by some nonneutral synthetic surfactants as a result of surfactant-induced permeabilization of the cells... 

Once proteins are transferred into the periplasm they can also be excreted into cultivation media (but with further reduction of the yield). This mainly happens by leakage, which can be enhanced by osmotic shock or cell wall permeabilization (by mutations or enzymatically). However, in some cases passive excretion does not work. There are also strategies with transplantation of active transport proteins for one or both of the membranes [35], but so far they are not that reliable. 

Some cell lines, such as HEK293, may detach during the permeabilization step due to a strong Ca2+ dependence for attachment. While it is critical that cells are permeabilized as a monolayer, detachment does not seem to hinder cytosolic ribosome release, as they tend to detach as a (partial) monolayer. Following the permeabilization step, cells can simply be separated from the soluble cytosol phase by centrifugation at 750-1000 Xg for 5 min. Transfer the supernatant (cytosol) to a new tube. Remove any remaining cells attached to the flask via the wash buffer, combine with the cell pellet, and recover by centrifugation. Proceed with the membrane solubilization step. 

Adam, SA., Marr, R. S., and Gerace, L. (1990). Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. Ill, 807—816. 

Comments Permeabilization can also be done with 0.1 to 0.3% Triton X-100 or NP-40 for 5 min at RT. Permeabilization methods should be optimized for different antibodies and preservation of different subcellular structures (Allan, 2000). 

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