Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n-3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H+ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n-3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n-3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.

Background-A new intracellular adenosine triphosphate (ATP) delivery technique has been developed and was tested for skin wound care.

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Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N2 to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [ 14 C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes. The bacterium Bacillus subtilis is a Gram-positive sporeforming rod that has been used extensively in molecular biology and biotechnology. The bacterium has a relatively simple cell wall consisting of approximately equal amounts of peptidoglycan and teichoic acid. During a normal doubling, 50% of the wall is turned over into the culture medium each generation and is not reused for growth (1, 2). Turnover is restricted to the outermost cylindrical peptidoglycan, or wall that is at least one generation old (3, 4). The excision of the peripheral wall is brought about by the action of the autolysin N-acetyl-muramoyl-L-alanine amidase. All bacteria are thought to possess autolysins (5), yet surprisingly very little is known about the regulation of the enzymes.It was observed by Jolliffe et al. (6) that any condition imposed on B. subtilis leading to dissipation of the protonmotive force (pmf) caused the bacteria to autolyze, suggesting the energized membrane was coupled to autolysin regulation. Agents, such as azide and carbonylcyanide-m-chlorophenylhydrazone, initiated autolysis of B. subtilis. In fact, any condition that prohibited carbon metabolism, including anaerobiosis and starvation, seemed to promote autolysis. Kemper et al. (7) speculated that during carbon utilization, secreted protons could neutralize the negative charges in the cell wall, resulting in a relatively low pH surrounding the protoplast. This acidic pH would prevent autolysin activity, thus providing a means for autolysin inhibition. By use of pH-sensitive probes and selective chemical...

Docosahexaenoic acid (DHA), a long-chain polyunsaturated omega 3 fatty acid, is tested to determine its mode of action as an anti-cancer agent. We demonstrate that DHA can increase the permeability of phospholipid vesicles, as monitored by vesicle swelling in isosmolar erythritol and leakage of sequestered carboxyfluorescein, and T27A tumor cells, as monitored by swelling in isosmolar erythritol and release of sequestered 51Cr. DHA was incorporated into lipid vesicles as either the free fatty acid or as 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine. DHA was incorporated into the tumor cells by fusion with vesicles made from the mixed-chain phosphatidylcholines. DHA is demonstrated here to be much more effective in increasing permeability than is oleic acid, the major unsaturated fatty acid normally found in tumor plasma membranes. It is proposed that incorporation of DHA makes tumor plasma membranes substantially more permeable, which may explain, in part, its anti-tumor properties.

alpha-Thrombin, bradykinin, and histamine are endogenous mediators that increase endothelial permeability. We examined the mechanism by which these three vasoactive mediators could alter permeability to albumin of human umbilical vein endothelial cells (HUVEC). HUVEC were grown to confluence on Transwell membranes and we monitored the flux of fluorescein isothiocyanate-labeled human serum albumin across the membrane from the upper to lower chamber of the Transwell. Addition of alpha-thrombin, bradykinin, or histamine increased the permeability coefficient of the HUVEC monolayer. At 30 min the permeability coefficient for alpha-thrombin was 4.92 x 10(-6) cm/sec while histamine was 4.47 x 10(-6) cm/sec. Maximum changes in the permeability coefficient were about three-fold control baseline values (1.59 x 10(-6) cm/sec). There was also a temporal difference in the magnitude of the permeability coefficient. alpha-Thrombin and bradykinin induced HUVEC permeability was increased for the first 90 min after which it returned to control levels. In contrast, histamine increased the permeability of the HUVEC monolayer throughout the 2 h experiment. To determine a possible intracellular mechanism of the altered permeability coefficients, HUVEC were labeled with FURA-2 and intracellular calcium was monitored by digital fluorescence ratio imaging. Maximum intracellular calcium in HUVEC was increased by alpha-thrombin (245 +/- 20 nM) and histamine (210 +/- 22 nM), but not by bradykinin (70 +/- 7 nM) as compared to control (69 +/- 10). Fluorescent photomicrographs of HUVEC stimulated with the three agonists indicated that alpha-thrombin and histamine substantially altered HUVEC f-actin arrangement, while bradykinin had no effect on HUVEC f-actin distribution. These data support previous in vitro and in vivo studies demonstrating increased permeability by all three agonists. These data also show, for the first time, that histamine and alpha-thrombin increased permeability by calcium-dependent intracellular pathways, but bradykinin operates through a calcium-independent mechanism.

Optimal curcumin delivery for medicinal applications requires a drug delivery system that both solubilizes curcumin and prevents degradation. To achieve this, curcumin has been encapsulated in submicrometer chitosan/Tween 20 particles via a benchtop spray-drying process. Spray-drying parameters have been optimized using a Taguchi statistical approach to minimize particle size and to favor spheroid particles with smooth surfaces, as evaluated with scanning electron microscopy (SEM) imaging. Nearly spherical particles with 285 ± 30 nm diameter and 1.21 axial ratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within the chitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 h period.