Intact lipids

Ambrose (1990, 1993) found that for most samples well-preserved collagen had at least 3% carbon and 1% nitrogen. If this criterion is applied in this study, two of our 22 samples show aberrant values for carbon and one of 22 is aberrant for nitrogen, with three of the samples containing 1% nitrogen (Table 7.1). Of the three samples where we can compare bone with hpid-removed and lipids intact, both C and N concentrations are within acceptable limits in the lipid-removed sample, but substantially lower in most layers with lipid intact. The concentrations of both C and N are low, but within acceptable criteria, in the sample with well preserved histology (C = 5.11%, N= 1.77%). 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

Using the reconstitution approaches described above, we have demonstrated that phosphorylation of the skeletal muscle Ca channels by PKC results in activation of the channels [108], In the fluo 3-containing liposomes, channels phosphorylated by PKC exhibited a two-fold increase in the rate and extent of Ca " influx [108], Using the lipid bilayer-T-tubule membrane reconstitution system we are currently analyzing the effects of PKC-catalyzed phosphorylation at the single channel level [133], The demonstration that these channels undergo phosphorylation as a result of activation of PKC in intact skeletal muscle cells has not yet been achieved. 

Moen MA, KE Hammel (1994) Lipid peroxidation by the manganese peroxidase of Phanerochaete chrysosporium is the basis for phenanthrene oxidation by the intact fungus. Appl Environ Microbiol 60 1956-1961. 

The reaction was started by transferring 1 mL of the enzyme/buffer/bile salt solution (pH=7.2, 37 C) to each flask placed in a thermostated shaker at 37°C. Experiments were carried out without lipid and bile salt as well, and in these experiments equal amounts of stock solutions of the enzyme in buffer and peptide in buffer were mixed in the flasks at time zero, to give the indicated concentrations (see Table III). The reactions in the flasks were stopped by adding 0.5 ml acetonitrile at different times. The total amount of intact peptide remaining in a flask was determined by HPLC, after the content was dissolved by adding ethanol. 

Gorter and Grendel in 1925 [527], drawing on the work of Langmuir, extracted lipids from RBC ghosts and formed monolayers. They discovered that the area of the monolayer was twice that of the calculated membrane surface of intact RBC, indicating the presence of a bilayer. This was the birth of the concept of a lipid bilayer as the fundamental structure of cell membranes (Fig. 7.1). 

The introduction of fast-atom bombardment (FAB) half a decade later extended the analyst s biomarker repertoire to intact polar lipids, desorbed... 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Jones, J. J. Stump, M. J. Fleming, R. C. Lay, J. O. Wilkins, C. L. Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS. /. Am. Soc. Mass Spectrom. 2004,15,1665-1674. 

Unheated carrot juices produced from carrots blanched at 80°C for lOmin were devoid of di-isomers, and further pasteurization or sterilization process formed only 13-d.v-P-carotene, respectively, at 2% and 5% (Marx et al. 2003). However, extensive carrot blanching (100°C for 60min) caused the losses of 26%-29% in total P-carotene content, along with an increased 13-d.s-P-carotene content up to 10% after pasteurization (Tmax 95°C, F = 3) and to 14% after sterilization (Tmax 121°C, F = 5) (Marx et al. 2003). The addition of grape oil to carrot juice before heat treatment enhanced the 13-di-P-carotene formation (18.8%) as compared to the control (6.0%) (Marx et al. 2003). This fact is probably due to the partial dissolution of crystalline carotene, present in the intact carrot in lipid droplets, since the solubilization of carotenes during blanching is a prerequisite for the formation of di-isomers. 

P-gp substrates are in general either neutral or cationic at physiological pH (weak bases). Weak bases can cross the lipid membrane in the uncharged form and reprotonate in the negatively charged cytosolic leaflet of the membrane. With a few exceptions (e.g., the tetraphenyl phosphonium ion, which can reach the cytosolic membrane leaflet due to charge delocalization [70]), permanently charged cations do not cross the cell membrane and therefore cannot interact with P-gp in intact cells. They can, however, insert into the cytosolic leaflet in inside-out cellular vesicles and are then transported by P-gp [42, 71]. 

Fig. 8.4 TEM images showing ethylenediami-nopropyl-functionalized magnesium phyllosi-licate microtubules produced by lipid helicoid templating. In each case the template was removed by washing with ethanol. (A) intact...

Chlorella zofingensis cells are transferred to a growth medium, with a low nitrogen concentration (10% of normal concentration). After approximately 6-8 wk they develop a red colour, due to the decomposition of chlorophylls and synthesis of secondary carotenoids (stored in lipid droplets within the cytoplasm of the cells). At this stage the chloroplast are intact, although the surface area of thylacoids is mostly reduced. 

Activation of PE residues with these crosslinkers can proceed by one of two routes the purified PE phospholipid may be modified in organic solvent prior to incorporation into a liposome, or an intact liposome containing PE may be activated while suspended in aqueous solution. Most often, the PE derivative is prepared before the liposome is constructed. In this way, a stable, stock preparation of modified PE may be made and used in a number of different liposomal recipes to determine the best formulation for the intended application. However, it may be desirable to modify PE after formation of the liposomal structures to ensure that only the outer half of the lipid bilayer is altered. This may be particularly important if substances to be entrapped within the liposome are sensitive or react with the PE derivatives. 

Since the protein-palmitate derivative can t be dissolved in organic solvent during homogenization of lipid to form liposomal membranes, it must be inserted into intact liposomes by detergent dialysis. 

Intact biological membranes are far more complex systems than even the lipid extracts (see Fig. 6). In hardly any case do the lipid components exceed 50% of the dry weight, as membranes intrinsically contain numerous proteins and various glycoconjugates. This diversity is most pronounced in the plasma membrane (irrespective of the genera) which is effectively amalgamated with the non-lipidic... 

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