Permeabilised cells

Fluorescently labelled antibodies can be used to visualise cellular or subcellular structures. This is done by incubating antibodies against specific cellular antigens with frozen or fixed tissues sections, or even permeabilised cells (Javois 1994). Unbound antibodies are removed by washing, and then a second anti-immunoglobulin antibody coupled to a fluorescent group, such as fluorescein or rhodamine, is added to the preparation. The sample is washed free of excess fluorescent antibody and visualised using a fluorescence microscope. 

Before precise questions can be asked at the molecidar level about the important interactions of ADPRT with its substrates and with chromatin, further information is required concerning the response of the enzyme to stimuh under more nearly physiological conditions. To this end we have undertaken studies of the kinetics of ADPRT in permeabilised cells and have attempted to analyse the response of the enzyme to DNA strand breaks. 

The measurement of ADP-ribosyltransferase activity in situ, either in nuclei or in permeabilised cells is a procedure simple in execution but fraught with difficulties of interpretation. The incorporation of radioactivity from labelled NAD into TCA insoluble products represents at best the net rate of ADP-ribosylation [ADPRT activity less poly(ADP-ribose) gjycohydrolase and ADP-ribosylprotein lyase activities]. At zero time we may hope that the degradative enzymes, having no radiolabelled substrate, do not contribute to the rate. 

We have attempted to dissect the incorporation of NAD into these two reactions by snake venom phosphodiesterase (SVPDE) digestion. The rate of incorporation of label into 5 -AMP gives an estimate of the rate of initiation and the rate of incorporation into phosphoribosyl-AMP(PR-AMP) estimates the rate of elongation. AE-cellulose chromatography can be used to monitor the proportion of 5 -AMP deriving from mono- and poly(ADP-ribose) residues respectively. This provides an important check since, in permeabilised cells, mono(ADP-ribose) residues can be produced other than in the nucleus and also by non-enzymic reactions [9],... 

Kinetic Analysis of ADPRT Activity in Permeabilised Cells... 

Preparation of Cells. Assays for DNA damage (and Ho33342 uptake) were carried out on cultures which had been subjected to a standard protocol for the generation of freeze/thawed (permeabilised) cells directly from monolayer, adapted [17, 18] from the method described by Ganesan et al. [19]. Cells were resuspended in a low salt (LS) buffer (10 mM Tris-HCl, pH 8 100 mM NaCl 10 mM EDTA 1 mg ml bovine serum albumin). 

DNase II Nicking. Permeabilised cells (8 x 10 ) were pelleted in a series of... 

To assess whether the results obtained with Ho33342 related to some innate feature of cellular chromatin, namely accessibility, permeabilised cells were exposed to a low level of DNase II (under conditions which produced a linear relationship between enzyme concentration and break frequency) and accessibility monitored as the frequency of enzyme-induced DNA strand-breaks. The results (Table 1) show that nuclease sensitivity correlated with the frequency of ligand-induced breaks. 

Wilson GL, Dean BS, Wang G, et al. Nuclear import of plasmid DNA in digitonin-permeabilised cells requires both cytoplasmic factors and specific DNA sequences. J Biol Chem 1999 274 22025-22032. 

The elimination of the amino donor, L-aspartic acid, resulted in an almost complete reduction of activity. Neither cell permeabilisation nor cofactor (pyridoxalphosphate) addition were essential for L-phenylalanine production. Maximum conversion yield occurred (100%, 22 g r) when the amino donor concentration was increased. Aspartic add was a superior amino donor to glutamic add 35 g l 1 was used. 

A very efficient and universal method has been developed for the production of optically pue L- and D-amino adds. The prindple is based on the enantioselective hydrolysis of D,L-amino add amides. The stable D,L-amino add amides are effidently prepared under mild reaction conditions starting from simple raw materials (Figure A8.2). Thus reaction of an aldehyde with hydrogen cyanide in ammonia (Strecker reaction) gives rise to the formation of the amino nitrile. The aminonitrile is converted in a high yield to the D,L-amino add amide under alkaline conditions in the presence of a catalytic amount of acetone. The resolution step is accomplished with permeabilised whole cells of Pseudomonas putida ATCC 12633. A nearly 100% stereoselectivity in hydrolysing only the L-amino add amide is combined with a very broad substrate spedfidty. 

Shepherd VA, Goodwin PB. The porosity of permeabilised Chara cells. Aust J Plant Physiol 1989 16 231-239. 

Recent results indicate that not only topogenic signals and membrane composition contribute to the proper topology of a membrane protein. The antimicrobial peptide nisin, produced by Lactococcus lactis, kills Gram-positive bacteria via pore formation, thus leading to the permeabilisation of the membrane. Nisin depends on the cell-wall precursor Lipid II, which functions as a docking molecule to support a perpendicular stable transmembrane orientation [43]. 

The diterpenes ferruginol and hinokiol [104, 105], geranylgeraniol, tepre-none and phytol [106] showed antibacterial activity. iS-Hydroxykaurenoic acid produced permeabilisation of the cell membrane of the fungi Botrytis cinerea [107, 108]. 

Many cells are susceptible to the appreciable shearing forces that arise on repeated freezing and thawing, or to hypotonic buffers which cause cells to swell up, and in certain cases to lyse this is particularly the case for cells in soft plant and animal tissue. Such treatments only rarely lead to complete cell lysis, the exceptions to this being erythrocytes and reticulocytes which are lysed quantitatively under hypotonic conditions. Non-mechanical homogenisation is of particular relevance to cells like yeast which are refractory to other procedures. One of the simplest procedures for yeast, which can certainly not be described as gentle, is toluene-induced autolysis. This is carried out at room temperature and leads to permeabilisation of the cell walls this causes various hydrolases to be activated causing breakdown not only of the cell structure, but also (undesirably) of many sensitive proteins and nucleic acids in the cell. Consequently, this process is mainly of historical interest. 

Catala P, Parthuisot N, Bernard L, Baudart J, Lemarchand K, Lebaron P (1999) Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes application to viability assessment in waters. FEMS Microb Lett 178 219-226 Cools 1, D Haese E, Uyttendaele M, Storms E, NeUs HJ, Debevere J (2005) Solid phase cytometry as a tool to detect viable but non-culturable cells of Campylobacter jejuni. J Microbiol Meth 63 107-114... 

El Izzi A, Benie T, Thieulant M-L, Le Men-Olivier L, Duval J (1992) Stimulation of LH Release from Cultured Pituitary Cells by Saponins of Petersianthus macrocarpus A Permeabilising Effect. Planta Med 58 229... 

Robinson I. R. Burgoyne. 1991. Characterization of distinct inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive calcium stores in digitonin-permeabilised adrenal chromaffin cells. J. Neurochem. 56 1587-93. 

Fig. 2. Depletion of Mcl-l protein levels by antisense treatment. Neutrophils were reversibly permeabilised with streptolysin O and incubated with 20 iJuM carboxyfluorescein (CF), inverse antisense (Inverse) or antisense sequence 1 (AS) of chimeric oligodeoxynu-cleotides. Suspensions were incubated in either the absence (—) or presence (+) of GM-CSF. Western blots were analysed on aliquots of 10 cells, 4 h after permeabilisation. Actin (Ponceau S-stained blots) is shown to indicate equivalence of loading. Representative blot of 3 independent experiments. Antisense sequences and structures are given by Moulding et al. [28]. 

---