Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

Oil-in-water liposomal emulsions: characterization and potential use in vaccine delivery.

Emulsification of mineral oil by phospholipids donated by liposomes composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol, and lipid A by extrusion resulted in the formation of oil-in-water liposomal emulsions containing a substantial number of intact liposomes. Increasing the proportion of liposomes from 25 mM to 150 mM phospholipid and increasing the oil content from 2.5% (v/v) to 42.5% (v/v) changed the flow characteristics of the emulsions from fluid liquid-like to viscous. Likewise, the degree of stability of the emulsions was liposomal phospholipid concentration-dependent, ranging from partial emulsification in the range 25-100 mM to complete stabilization in the range 125-150 mM. Despite some loss of liposome integrity, as evidenced by the release of liposomal trapped glucose, emulsification of liposomes containing encapsulated prostate-specific antigen (PSA) exhibited antigen-specific immunostimulation in mice. These results suggest that liposomes containing encapsulated antigen can serve as constituents for the formulation of oil-in-water vaccines.

Is lateral phase separation required for fatty acid to stimulate lipases in a phosphatidylcholine interface?

Lipase-catalyzed oxygen exchange between 13,16-cis,cis-docosadienoic acid and water in liquid-expanded monolayers with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine exhibits abrupt, lipid composition-dependent changes in extent and mechanism [e.g., Muderhwa, J. M. and Brockman, H. L. (1992) J. Biol. Chem. 267, 24184-24192]. The critical nature of this transition suggests possible lateral phase separation of the lipids. This has been addressed by substituting for either lipid species one which can exist in more condensed monolayer states. Analysis of phase transition surface pressures as a function of lipid composition shows that each set of fatty acid-phosphatidylcholine mixtures exhibits a finite range of miscibility in liquid-expanded monolayers. These results strongly suggest that 13,16-cis,cis-docosadienoic acid and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine are miscible under the conditions of the oxygen-exchange experiments. Furthermore, to address more directly the relation of lateral lipid phase separation to lipase regulation, oxygen exchange catalyzed by pancreatic carboxylester and triglyceride lipases was studied using mixed monolayers of [18O]2-docosadienoic acid and 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine. These lipids are miscible in the liquid-expanded state at all compositions. The lipid composition dependencies of both the extent and mechanism of lipase-catalyzed oxygen exchange were essentially identical to those obtained earlier. Thus, lateral lipid phase separation is not required for the critical transition in substrate accessibility to lipases. This finding supports a percolation-based model of lipase regulation within a single surface phase and suggests the "topo-temporal" regulation of lipid-mediated signaling in cells.

Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.

Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 2. Effects of lateral lipid distribution in mixtures with phosphatidylcholine.

The lipase-catalyzed exchange of the carboxyl oxygens of 13,16-cis,cis-docosadienoic acid (DA) was studied in the presence of a nonsubstrate matrix lipid, 1-palmitoyl-2-oleoylphosphatidylcholine. For mixed lipid films at the argon-water interface exposed to pancreatic carboxylester lipase (EC 1.1.1.13), the extent of oxygen exchange showed an abrupt increase as the abundance of DA in the interface was increased from 0.5 to 0.6 mole fraction. This compositional range was independent of the level of enzyme used and of the surface pressure, i.e., lipid packing density, of the film. Concomitant with the transition was a change in the apparent mechanism of exchange from coupled to random sequential. Like the extent of oxygen exchanged, the shift in mechanism was independent of all variables except the lipid composition of the interface. The absence of any chemical or physical change accompanying the exchange reaction precludes mechanistic explanations based on the generation of reaction products by the enzyme. Instead, the results suggest that the lateral distribution of DA in phosphatidylcholine-DA interfaces regulates the expression of carboxylester lipase activity and its apparent mechanism. Preliminary measurements give an average cluster size of 1825 molecules of DA when its mole fraction is 0.35. As the DA content of the interface reaches 0.5-0.6, there appears to be a lipid head-group based percolative transition in which DA becomes the continuum. Because this transition involves the lateral organization of the lipids themselves, other interfacially active enzymes may be regulated similarly.

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.

Binding of pancreatic carboxylester lipase to mixed lipid films. Implications for surface organization.

The hydrolysis of ester substrates in mixed monolayers with 1-palmitoyl-2-oleoylphosphatidylcholine by pancreatic carboxylester lipase (CEL) shows an abrupt increase from less than 10 to 100% when substrate abundance exceeds a critical value (Tsujita, T., Muderhwa, J.M., and Brockman, H.L. (1989) J. Biol. Chem. 264, 8612-8618). Adsorption of native CEL to these mixed lipid films under the same conditions shows a monotonic increase with substrate abundance to a maximum of approximately 4 pmol/cm2. In the range of low substrate hydrolysis, bound enzyme reaches 0.6 pmol/cm2 or about one-sixth of an enzyme monolayer. Given the high turnover number of the enzyme, absence of bound catalyst cannot explain the lack of substrate hydrolysis. Similarities of the adsorption data obtained with different substrates suggest a common, area-based mechanism with an excluded area for the phospholipid. Quantitative analysis confirms this and gives an excluded area of 43.5 +/- 1.0 A2/molecule. Comparable studies with the hydrolysis product, oleic acid, replacing each substrate show markedly different results. Up to oleic acid mol fractions of 0.5, bound CEL levels are essentially constant at 0.215 pmol/cm2. This behavior relative to substrate-containing films cannot be ascribed to differences in surface pressure, surface potential, monolayer compressibility, nonexcluded area, CEL denaturation, or oleic acid charge. Together with the ultrasensitive composition dependence of substrate hydrolysis, the data suggest that phospholipid-based organization of the surface is a primary regulator of enzyme binding and catalysis.

Lipid-lipid interactions as regulators of carboxylester lipase activity.

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.