Mechanisms for the interaction of the milk fat globule membrane with the plasma membrane of gut epithelial cells.

The milk fat globule membrane (MFGM) provides infants and adults with several health benefits. These are not derived solely from its unique composition, but also from arrangement of lipids in the MFGM that, in the case of newborns, could reach the intestine partially intact. Fluorochromes associated with lipid derivatives were used to prove a fusion process between the MFGM and the cellular membrane of differentiated Caco-2 cells. To explore the mechanism of this interaction, incubations of MFGM with Caco-2 cells were carried out in the presence of fusogenic agents or compounds that block other MFGM interaction pathways with cells. Confocal fluorescence microscopy provided visual evidence of the fusion process. Lastly, determination on the lipid profile of cells after their interaction with MFGM indicated a metabolic rearrangement of lipids leading to accumulation of triacylglycerols.

A colorimetric method for determination of γ-glutamyl-S-ethenyl-cysteine in narbon vetch (Vicia narbonensis L.) seeds.

A new colorimetric method based on the bleaching of the iodoplatinate ion has been developed for fast and easy determination of γ-glutamyl-S-ethenyl-cysteine (GEC) in narbon vetch (Vicia narbonensis L.) seeds. The calibration curve showed a good correlation (r(2)=0.9959) between absorbance and GEC amounts from 5.5 to 33 µg (10-59.78 µmol/L). The limits of detection and quantification were 1.16 and 3.55 µmol/L, respectively, and no significant interferences from other sulfur-containing compounds were observed. The method showed excellent repeatability (relative standard deviation [RSD]=0.28%), reproducibility (RSD=4.4%), and accuracy (94%). Determination of GEC in 20 narbon vetch accessions yielded values that were in agreement with those reported previously using capillary electrophoresis and high-performance liquid chromatography methods. The method could be especially valuable for determination of GEC during the process of production of new low-GEC narbon vetch varieties.

Phospholipase D and priming of the respiratory burst by H(2)O(2) in NR8383 alveolar macrophages.

Previous investigation showed that preincubation within a range of nontoxic H(2)O(2) concentrations enhanced subsequently stimulated superoxide production by rat alveolar macrophages in response to various stimuli. In the present study, the NR8383 rat alveolar macrophage cell line was used to further investigate the priming effect of H(2)O(2). Using nitroblue tetrazolium, which formed an insoluble formazan when reduced by superoxide, modulation of the respiratory burst was visualized in a cell population exposed to a concentration gradient of H(2)O(2) before stimulation. This model system illustrates how H(2)O(2) may constitute a signaling molecule for a feed-forward regulation of the respiratory burst during inflammation. n-Butanol, which allows consumption of phosphatidic acid by the transphosphatidylation reaction, and propanolol, which inhibits phosphatidic acid phosphohydrolase, were used to investigate the possible involvement of phospholipase D in this phenomenon. These two agents were found to inhibit the basal adenosine diphosphate-stimulated respiratory burst. Inhibition of the H(2)O(2)-enhanced respiratory burst was equally or slightly less effective when expressed as percentage of controls. Furthermore, phospholipase D was not activated by H(2)O(2) concentrations that enhance superoxide production. Thus, phospholipase D does not mediate the enhancement of the respiratory burst by H(2)O(2), although it may be activated by high concentrations of this hydroperoxide.

The alveolar macrophage as a model of calcium signaling in oxidative stress.

Regulation of the free intracellular calcium concentration, [Ca2+]i, plays a major role in physiological signal transduction. Many of the essential enzymes in signaling cascades are Ca(2+)-dependent, as are numerous proteins that participate in the regulated function. Oxidative stress, which for many years was considered synonymous with cell and tissue injury, has more recently been demonstrated to alter signal transduction in both positive and negative directions. The realization that hydrogen peroxide and lipid hydroperoxides are produced as part of normal metabolism has led to the proposal that these oxidants function as second messengers. Exposure to environmental and other agents that produce hydroperoxides or the addition of exogenous hydroperoxides also causes elevation of [Ca2+]i in some cells. At sublethal exposure to hydroperoxides, the elevation in [Ca2+]i can either alter or mimic physiological stimulation. In addition to endoplasmic reticulum, mitochondria, and the extracellular space, the phospholipid- and Ca(2+)-binding proteins known as annexins constitute a Ca2+ pool from which this ion may be released under situations of oxidative stress. In this article, the source and consequences of Ca2+ elevation are reviewed with an emphasis on studies done with alveolar macrophages. These phagocytes, which modulate much of the physiological and immunological function of the lung, are susceptible targets for environmental oxidants.

Effects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes.

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe3+, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by 18O incorporation from H2 (18)O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE. TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.

Stimulation of hepatocyte glycerolipid synthesis by iron/ADP is due to ADP rather than oxidative stress.

ADP-complexed Fe3+ has been used in various in vitro systems and in intact cells to induce lipid peroxidation. During studies on the effects of oxidative stress on lipid metabolism we observed a significant increase in de novo glycerolipid synthesis in Fe3+/ADP-treated rat hepatocytes as evidenced by increased [U-14C]glycerol incorporation. Here we show that this increase, largely due to enhanced triacylglycerol synthesis, is caused by ADP rather than Fe3+/ADP-induced oxidative stress. Hence, metabolic alterations due to treatment of intact cells by Fe3+/ADP must be interpreted with caution.

Peroxidative modification of a membrane protein. Conformation-dependent chemical modification of adenine nucleotide translocase in Cu2+/tert-butyl hydroperoxide treated mitochondria.

Peroxidative treatment of rat heart mitochondria results in a gradual increase of the apparent molecular weight of the adenine nucleotide translocase (ANT) by up to 1.2 kDa. ANT isolated from mitochondria treated with 1 mM tert-butyl hydroperoxide and 5-40 microM Cu2+ for 1 h at 37 degrees C exhibited a progressive loss of lysine, cysteine, arginine, and valine residues compared to native ANT. N-Ethylmaleimide, dithiothreitol, and the specific inhibitor of ANT, carboxyatractyloside (CAT), inhibited the peroxidation-induced molecular weight shift without inhibiting lipid peroxidation, which is believed to be the primary cause of the observed ANT modification. Bongkrekic acid, which stabilizes ANT in a conformation different from that brought about by CAT, did not inhibit the ANT molecular weight shift. Dithiothreitol, as well as CAT, was found to protect ANT against most of the losses of amino acid residues, indicating that alteration of sulfhydryl residues is required for chemical modification of, not only cysteine, but also lysine, arginine, and valine. We conclude that the peroxidative modification of ANT is conformation-dependent and involves chemical modification of cysteine as a critical step.

Peroxidative damage to cardiac mitochondria. II. Immunological analysis of modified adenine nucleotide translocase.

We have previously reported that treatment of isolated rat heart mitochondria with the free radical-generating system Cu2+/tert-butylhydroperoxide produces striking changes in the adenine nucleotide translocase (ANT) of the inner membrane. These changes include a small increase in apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by its disappearance from the polypeptide profile upon further oxidant treatment (Zwizinski and Schmid (1992) Arch. Biochem. Biophys. 294, 178-183). In order to characterize its peroxidative modification in more detail, we have purified rat heart ANT and prepared polyclonal antibody against it. Using this antibody, we have observed that increasing oxidant treatment results in a gradual increase in the ANT protein's apparent molecular weight by up to 1 kDa. The progressive nature of the molecular weight shift, which parallels the generation of thiobarbituric acid reactive substances, supports the hypothesis that this phenomenon may be the result of covalent addition of increasing amounts of lipid peroxidation products. Strong oxidative treatment of cardiac mitochondria also causes fragmentation and polymerization of the ANT protein. However, Western blot analysis showed that a major portion of the original ANT survives even extensive oxidation as a distinct, modified protein. Therefore, the almost complete disappearance of ANT from Coomassie-stained gels appears to be the result of cross-linking and fragmentation reactions, as well as a decreased efficiency of the Coomassie staining. Because a measurable molecular weight shift of ANT occurs at the mildest oxidative treatment tested (resulting in the production of only 1.1 nmol malondialdehyde/mg protein), it may be relevant as a parameter of myocardial ischemia-reperfusion injury.