Results outer membrane preparation

It is possible to permeabilize the outer membrane of normal cells (with detergent or alcohol) in order to allow propidium iodide to enter the nuclei. If we then treat the normal cells with RNase in order to ensure that any fluorescence results from their DNA content (without a contribution from double-stranded RNA), we find that the nuclei fluoresce red with an intensity that is more or less proportional to their DNA content. By the use of a red filter and a linear amplifier on the photomultiplier tube, we can detect that red fluorescence. The channel number of the fluorescence intensity will be proportional to the DNA content of the cells. The method is simple and takes about 10 minutes. Flow cytometric analysis of the red fluorescence from the particles in this preparation of nuclei from normal, nondividing cells will result in a histogram with a single, narrow peak (see the first histogram in Fig. 8.1) all the particles emit very nearly the same amount of red fluorescence. This supports our knowledge that all... 

While most workers report the outer membrane siderophore receptors to have molecular weights in the 75-95K range, some variation in the magnitude of these numbers may be attributed to the preparative and analytical methods as well as to the particular standards used. Since enterobactin will rapidly remove iron from ferrichrome, the transport of the latter must perforce be studied in mutants lacking the former. However, such mutants often display multiple lesions. Additionally, isogenic strains have seldom been used and variations in media and cultural conditions will further confound attempts to compare results reported from different laboratories. 

Direct fluorescent antibody smears have become a more efficient method than Giemsa stains or tissue cultures fiar identifying chlamydia. Commercially prepared kits make specimen collection convenient, and results are available in approximately 24 hours. Good results, however, depend on obtaining an adequate specimen. Fluorescein-labeled monoclonal antibodies in the staining reagent specific for Chlamydia trachomatis outer membrane proteins bind to the C. trachomatis in the smear. Studies that compare direct fluorescein antibody techniques with tissue culture results have found acceptable sensitivity and specificity values. 

Mitochondria have been prepared from plant tissues by a number of different techniques. Unfortunately, the variations in technique may also be reflected in the somewhat different compositions reported. Mitochondria, in general, have membranes containing a high concentration of phospholipids. Phosphatidylcholine and phosphatidylethanolamine are major components and are fairly evenly apportioned between the inner and outer membranes. Cardiolipin is found in high amounts in the inner membrane, while phospha-tidylinositol is mainly in the outer membrane. Phosphatidylglycerol is a minor component. These results are illustrated in Table XV. 

We prepared sandwich type of laminates by using different type of PC based membranes as the outer membrane. The inner membrane was the PES membrane which was described in the previous section. The enzyme layer prepared by using 6 il solution of GOD with BSA and 3 pi of glutaraldehyde (5%). The results of the studies related to the optimization of the composition of the enzyme layer were given elsewhere in detail (7.. We used these laminates with die Rank electrode described above, and measured the electrode response of the glucose in buffer solutions (5 ml) containing different amounts of glucose (up to 100 mM). 

The actual steps involved in the fabrication of the sensors have been described elsewhere (18,19), The amperometric sensor onto which the glucose oxidase and outer membrane are deposited was fabricated using thin/thick film processing techniques. This process resulted in a three-electrode sensor with a 0.1 mm platinum-black working electrode. The sensor also contained a platinum-blacked counter electrode and a Ag/AgCl reference electrode. The preparation, deposition, and characterization of the outer membrane and glucose oxidase layers will be discussed here. 

While it is reasonable to assume that a significant portion of the cytoplasmic ribosomes which remain attached to purified mitochondria are those visualized by electron microscopy as attached to the outer mitochondrial membrane in situ, the extent to which adventitious association of cytoplasmic polysomes with the outer mitochondrial membrane occurs during cell fractionation is difficult to evaluate quantitatively. As an approach to this problem we determined conditions which would completely eliminate polysome binding to mitochondria in vitro, and then determined what effect these conditions had on the recovery of cytoplasmic rRNA with purified mitochondria. We showed that the binding of bound 80 S polysomes or free cytoplasmic polysomes to preparations of EDTA-washed mitochondria is completely inhibited by either 350 mM KCl or 10 m aurintricarboxylic acid (ATA). We found that when cells were opened and fractionated in buffer containing either 350 mM KCl or 10 M ATA, there was no significant reduction in the recovery of cytoplasmic rRNA with mitochondria when compared to mitochondria isolated in the absence of these components. These results support the notion that ribosomes which are attached to the outer mitochondrial membrane in isolated mitochondrial preparations are those which are seen attached to the outer membrane in situ. 

In another study, Fritzsche et al. span polysulfone hollow fiber membranes from solvent mixtures of N-methyl pyrrolidone (NMP)/formamide (FA), NMP/acetic acid (AA), NMP/propionic acid (PA), NMP/butyric acid (BA) and NMP/isobutyric acid (IBA). Results from oxygen plasma-etching experiments and SEM showed that the skin structure of the membrane prepared from the aliphatic acid (C2-C4) NMP complexes composed of aggregations of spherical nodules that became more loosely packed further away fi om the outer surface layer. Pores and channels were also observed but their dimensions decreased due to the resolution of the SEM. 

Another significant component of many liposome preparations is cholesterol. In natural cell membranes, cholesterol makes up about 10—50% of the total lipid on a molar basis. For liposome preparation, it is typical to include a molar ratio of about 50% cholesterol in the total lipid recipe. The addition of cholesterol to phospholipid bilayers alters the properties of the resultant membrane in important ways. As it dissolves in the membrane, cholesterol orients itself with its polar hydroxyl group pointed toward the aqueous outer environment, approximately even, in a three-dimensional sense, with the glyceryl backbone of the bilayer s phosphodiglyceride components (Fig. 337). Structurally, cholesterol is a rigid component in membrane construction, not having the same freedom of movement that the fatty acid tails of... 

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