Semi-intact cell

High-throughput, Permeable cell. Semi-intact cell... 

Beckers, C. J., et al. 1987. Semi-intact cells permeable to macromolecules use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell 50 523-534. 

Walaschewski, U., Jans, D. A.. Neugebauer, D. C.,and Peters, R. (1995). Semi-intact cells for nuclco-cytoplasmic transport studies. Methods Mol. Cell. Biol. 5, 87-95. 

Miller, S. G., and Moore, H.-P. H. (1991) Reconstitution of constitutive secretion using semi-intact cells Regulation by GTP but not calcium. /. Cell Biol. 112, 39-54. 

After aspirating the HKM buffer, 5 ml of ice-cold HES buffer is added to the dish. The cells are scraped off with a rubber cell-Ufter and the resulting cell suspension is transferred into a 15-ml Falcon tube (BD Biosciences, Franklin Lakes, NJ). This process is repeated once to ensure maximum recovery of the scraped cells. Subsequently, the cells are recovered by centrifugation at 750g for 5 min at 4° and resuspended in 150 p of ice cold HES buffer by pipetting up and down 10 times using a blue tip. Typically, 180-200 p of semi-intact cells with a protein concentration of 1.5-4 pgJpX is obtained. The semi-intact cells are stored at -70° if not used immediately. 

Hela cells are sulfate-starved and incubated with STxB at 18° to accumulate STxB in the EE/RE (synchronization). Then the plasma membrane is selectively perforated with SL-0 and semi-intact cells are harvested. EE/ RE-TGN transport is reconstituted in test tubes with the semi-intact cells supplemented with rat liver cytosol, ATP regeneration system, S-sulfate, and the reagents to be tested at 37°. STxB is specially engineered to contain two tyrosine sulfation sites. Therefore, the arrival of STxB to the TGN is monitored by its acquisition of S incorporation, as tyrosine sulfation is a... 

Camell, L., and Moore, H.P., 1994, Transport via the regulated secretory pathway in semi-intact PC12 cells role of intra-cistemal calcium and pH in the transport and sorting of secretogranin II. J. Cell Biol. 127, 693-705... 

Membrane proteins are difficult to work with. Solubilization in a semi-fiinctional form is possible with the aid of certain non-ionic detergents (3) the sheath of detergent molecules attached to the hydrophobic regions of these molecules compromise studies of interactions of membrane proteins. Thus, a method applicable to an intact cell or organelle may have some value. The system here involves membrane proteins (PBPs) and cytosolic proteins (most of the other MGPs) and thus demonstrates that such proteins and their interacting ligands can be accessed. 

The explanation of these NMR phenomena are complicated by the existence of two theories for the behaviour of water in tissue. These are represented by the membreme theory ( ) in which intracellular water is assumed to have the properties of ordinary liquid water, but that its solutes may be different from the extracellular solution due to the semi-permeable nature of the intact cell membrane. A later theory due to Ling (.7) suggested that intracellular water may be extensively structured due to the high concentration of fixed charges on macromolecules. 

Verleur, N., Hettema, E.H., van Roermund, C.W.T, Tal H.F. Wanders, R.J.A. (1997) Eur. J. Biochem. 2 , 657-661. Transport of activated fatty adds by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system. 

Photobleaching of reaction centers from semi-aerobically grown strains in isolated chromatophores or in intact cells indicated no difference in the amount of functional reaction center present in the RC" X case. 

Our original focus was on auxins and how they elicit growth in intact plants. Accordingly, we employed a semi-intact pea epicotyl system in which the plumule and hook were cut off to remove meristematic cells and the major supply of endogenous auxin, and the cut stump was painted with lanolin containing various additives. Our earlier findings with this system showing that auxin induces cellulase synthesis in vivo and in vitro have been reported [12]. 

One of the main advantages from the use of organotypic cultures (Fig. 3a) is that this type of preparation allows maintaining an intact or semi-intact architecture of the tissue, so that, for example, transfected cells are immediately spotted at low magnification and their locations can be easily tracked (Fig. 3b). If a fluorescent nuclear stain (DAPI) is applied, it is also possible— in our example— to map with good precision the individual layers of the cerebellar cortex (Fig. 3c), and/or to label live/dead cells with two color fluorescence (Fig. 3d). 

ERGs, the measurement of whole-retina extracellular electrical response to light stimuli. The on- and off-ERG transients provide a crude massed measurement of synaptic activity downstream from photoreceptor activation. Records can also be made with intracellular electrodes of single photoreceptor responses in the intact eye. More recently, research has focused on single-cell recordings from isolated or semi-isolated photoreceptors in vitro. 

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