Physiological stress response in free-living Amazonian caimans following experimental capture.

When captured, free-living crocodilians respond by hyperstimulation of the hypothalamic-pituitary-adrenal (HPA) axis, which triggers a cascade of downstream events of physiological stress. We examined these responses in two unstressed, and stressed Amazonian caimans, Caiman crocodilus and Melanosuchus niger. Plasma corticosterone levels increased in both stressed caiman species. In M. niger, the levels of this hormone increased 5.2-fold compared with the basal range values, while in C. crocodilus this was only 1.7-fold. After stress, M. niger needed more than 6 h to return its corticosterone levels to basal range values, whereas in C. crocodilus just 0.5 h was enough. Downstream events were characterized by an increase in glucose levels, which is associated with corticosterone increments. Excessive muscle activity resulted in increased plasma lactate content in both species. Lactate levels were also related to plasma calcium concentration, possibly due to the buffering capacity for preventing lactic acidosis. Clearance of excessive lactate load was faster in M. niger (0.5 h) than in C. crocodilus (more than 6 h). Although both caiman species respond in the same way to capture, the amplitude and duration of activation of the HPA axis are different. M. niger may be potentially more sensitive to acute stress than C. crocodilus. On the other hand, C. crocodilus needs more time to recover from the lactic acid load. Our experiment provides a useful diagnostic tool for management and conservation programs, as well as evaluating the impacts of tourism and recreational capture on caimans in the Amazon.

Protection by two complexing agents, thiosulphate and dissolved organic matter, against the physiological effects of silver nitrate to rainbow trout (Oncorhynchus mykiss) in ion-poor water.

Adult rainbow trout (Oncorhynchus mykiss) were exposed in ion-poor water ( approximately 50 microM Ca) to silver added as AgNO(3) or to AgNO(3) plus either thiosulphate (Na(2)S(2)O(3)) or dissolved organic matter (DOM). The effects of these exposures were assessed through repetitive blood sampling over 4 days. Trout exposed to 0.1 microM AgNO(3) alone accumulated large amounts of Ag on their gills and in their plasma, showed progressive losses of plasma Na and Cl, and had elevated concentrations of plasma glucose. In one set of exposures trout exposed to AgNO(3) alone also had increased cough rates, slightly higher ventilation rates, somewhat lower arterial oxygen tensions, and increased blood lactate concentrations. In contrast, trout exposed to 0.1 microM AgNO(3) plus 5 microM thiosulphate or 35 mg C l(-1) DOM accumulated less Ag on their gills and in their plasma, and showed no adverse ionoregulatory or respiratory effects due to Ag. These results demonstrate ionoregulatory and sometimes respiratory effects in fish exposed to ionic Ag(+) in ion-poor water, depending on water chemistry, and demonstrate the protective effects of synthetic and natural complexing agents through a reduction in the amount of ionic Ag(+) available to bind at the gills.

Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature.

Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol. The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-alpha in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles. Acute exposure to pharmacological levels of ethanol enhanced the function of PITP. Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes. Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer. Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces. The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane. Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles. Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids. The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action. In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid.

Saturable ethanol binding in rat liver mitochondria.

The binding of ethanol to rat liver mitochondria is shown to be saturable at physiologically relevant ethanol concentrations. This effect is reversible and is not observed in extracted mitochondrial phospholipids. Brief exposure of the mitochondria to heat abolishes saturable ethanol binding. Previously, saturable ethanol binding was reported in rat liver microsomes. Taken together, the studies indicate that saturable ethanol binding motifs may be widespread in cellular membranes. The possibility is raised that incomplete expression of the hydrophobic effect in membrane assembly results in the expression of amphipathic packing defects which display an affinity for and a sensitivity to ethanol. The presence of saturable binding modalities is reconciled with the long-standing consensus on the biodistribution of ethanol - that ethanol's interactions with tissue are negligible - on the grounds that the affinities of ethanol and of water for membranes are similar; consequently, free ethanol concentrations are insensitive to the presence of tissue despite significant ethanol binding. A fraction of the binding sites possess submillimolar affinities for ethanol consistent with published functional studies, both in vitro and in vivo, that reported submillimolar efficacies for ethanol.

Direct determination of hydration in the interdigitated and ripple phases of dihexadecylphosphatidylcholine: hydration of a hydrophobic cavity at the membrane/water interface.

Hydrophobic cavities at the membrane/water interface are stably expressed in interdigitated membranes. The nonsolvent water associated with 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (Hxdc(2)GroPCho) in the interdigitated (L(beta)I) and ripple (P(beta')) states and with its ester analogue 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (Pam(2)PtdCho) in the gel (L(beta')) and P(beta') states are determined directly. In the L(beta)I state at lower temperatures (4-20 degrees C), 16-18 water molecules per phospholipid are bound, consistent with water-filled cavities and hydrated headgroups. At 28 degrees C, the nonsolvent water decreases to 12, consistent with a reduction of the cavity depth by 0.34 nm due to increased chain interpenetration. This geometric lability may be a common feature of hydrophobic cavities. Only 5.4 waters are bound in the noninterdigitated P(beta') (40 degrees C), whereas the ester bound 8.1 waters in its P(beta') (37 degrees C), a difference of about one water per ester carbonyl. The relative dehydration of the ether linkage is consistent with it promoting more densely packed structures, which in turn, accounts for its ability to interdigitate.

Human sexuality in long-term care.

Sexual expression is a basic human need, a normal part of life that is integral to who we are as human beings. As we age, we continue to be challenged to grow in every area of our lives, including our sexuality. In institutionalized settings, however, human sexuality can be an area in which growth is not fostered, but restrained. Challenges leading to restraint include limited privacy, concerns about the consent of cognitively impaired sexual partners and conflicts with the personal values of institutional staff. Challenges exist in many areas, including inappropriate sexual expression toward a staff member or other residents.

Tracking phospholipid populations in polymorphism by sideband analyses of 31P magic angle spinning NMR.

A method was developed to track the distributional preferences of phospholipids in polymorphism based on sideband analyses of the 31P magic angle spinning nuclear magnetic resonance spectra. The method was applied to lipid mixtures containing phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and either cholesterol (Chol) or tetradecane, as well as mixtures containing the anionic phosphatidylmethanol, phosphatidylethanolamine, and diolein. The phospholipid composition of coexisting lamellar (Lalpha) and inverted hexagonal (HII) phases remained constant throughout the Lalpha --> HII transition in all mixtures, except those that contained saturated PtdCho and unsaturated PtdEtn in the presence of cholesterol-mixtures that are known to be microimmiscible because of favored associations between Chol and saturated acyl chains. In the latter mixture, saturated PtdCho was enriched in the planar bilayer structure, and unsaturated PtdEtn was enriched in the highly curved HII structure. This enrichment was coincident with an increase in the transition width. When compositional heterogeneity among coexisting phases was observed, it appeared that preexisting lateral microheterogeneities led to compositionally distinct transitional clusters, such that the distributional preferences that resulted were not those of the individual phospholipids.

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications.

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.

Elderly patients' experiences with nurses guided by Parse's theory of human becoming.

The purpose of this study was to describe the experience of being cared for by nurses who are guided by Parse's theory of human becoming from the perspective of hospitalized elderly patients. Open-ended interviews were used to collect data from a sample of 10 patients. The descriptions obtained from the interviews were analyzed using Colaizzi's phenomenological method. Three common themes constituted an exhaustive description of patients' experiences of relating to their nurses: coming together around instrumental tasks, nurses being there for patients, and nurses' pleasing way. These patterns of relating with nurses signify that patients felt both cared for and looked after. The findings suggest that Parse's theory of human becoming may provide one framework to ensure that caring takes place between nurses and their patients. The study has implications for nursing practice and research.

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol.

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.

Ethanol binding to a model carbohydrate, glycogen.

The binding affinity of ethanol for carbohydrates is unknown. Glycoconjugates are postulated to be sensitive targets of ethanol action. The glycogen content of muscle, liver, and brain is sensitive to ethanol. To explore whether carbohydrates as a class have a specific affinity to bind ethanol, we measured the binding of ethanol and other small molecules to the carbohydrate glycogen. Ethanol binding was found to be weak. The polar alcohol, glycerol, bound to glycogen with a greater affinity than ethanol did. Other small polar molecules (methanol, sucrose, acetate, glycine, and dimethyl sulfoxide) also bound more strongly than ethanol did. Ethanol and glycerol binding were concentration independent. No evidence of saturable or specific sites for these alcohols was obtained. Water binding was determined and was in agreement with hydrodynamic measures. Water binding exceeded the binding of all solutes studied. The loosely structured water of hydration in glycogen apparently was able to accommodate polar solutes, but tended to exclude ethanol and, to a lesser extent, methanol. We conclude that carbohydrates as a class exhibit no strong affinity or specificity for ethanol.

Saturable ethanol binding in rat liver microsomes.

The binding of ethanol to rat liver microsomes is shown to be saturable at clinically relevant ethanol concentrations, whereas this effect is not observed in extracted microsomal phospholipids. Brief exposure of the microsomes to heat abolishes saturable ethanol binding. Equilibrium binding data analysis, although only approximate in this context, suggests the presence of at least two groups of specific sites: high capacity sites with affinities near the pharmacological range and low capacity sites at lesser levels. The results indicate that the specificity of ethanol for tissue is considerably greater than previously recognized.

Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?

Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes.

Hydrophobic alkyl headgroups strongly promote membrane curvature and violate the headgroup volume correlation due to "headgroup" insertion.

The ability of lipid aggregates to form planar bilayers, rather than highly curved micellar or inverted structures, is dependent on the relative geometries of the headgroup and hydrocarbon regions. The headgroup volume approach to lipid structure provided a quantitative link between a lipid's headgroup size and its ability to promote curved, inverted hexagonal (H(II)) structures in a phosphatidylethanolamine (PtdEtn) matrix [Lee et al. (1993) Biophys. J. 65, 1429-1432]. Phosphatidylalkanols (PtdAlks) are shown here to promote curvature with a potency that far exceeds and a chain length dependence contrary to the expectations of the headgroup volume approach, suggestive of an atypical alkyl "headgroup" conformation. A homologous series of 3-substituted triacylglycerols (TAGs), for which 3-acyl "headgroup" insertion is established, exhibits a chain length dependence similar to the PtdAlks, evidence that the deviation is of common origin. The potency of the TAGs to promote curvature is unprecedented, and the onset of saturation, which parallels the dramatic promotion of curvature, occurs at mole fractions as low as 0.0025. The potency of the PtdAlks or TAGs to promote curvature exceeds that of all mammalian phospholipids examined. Thermodynamic analysis implicates the enthalpic curvature stress imparted upon the membrane matrix as the dominant energetic factor. The imparted stress ranges from -930 J mol(-1) for phosphatidylcholine to +7.5 kJ mol(-1) for 3-palmitoyl TAG. The results affirm the geometric considerations of membrane structure and indicate that alkyl headgroups tend to insert into the bilayer and increase the enthalpic curvature stress within the membrane.

Support for the shape concept of lipid structure based on a headgroup volume approach.

Headgroup volumes of seven dioleoyl lipid species, calculated from covalent radii, are shown to correlate linearly (r = 0.95) with the ability of those lipids to alter the midpoint temperature of the lamellar to inverted hexagonal phase transition (L alpha-->HII) of a 95 mole fraction percent phosphatidylethanolamine matrix. The results illustrate the utility of the shape concept and basic considerations of headgroup volume as a predictive tool for the determination of lipid structure.

Configurational entropy is the driving force of ethanol action on membrane architecture.

A colligative thermodynamic framework is developed to describe the action of ethanol on membranes. The partitioning of ethanol into a membrane structure imparts a randomness, configurational entropy, that stabilizes that structure from an energetic standpoint. When partitioning between membrane structures differs, the equilibrium between them is altered to favor the structure with the largest partition coefficient for ethanol. The action of ethanol and temperature originate in entropy and are equated through entropy. Membrane equilibria that are predicted to be most sensitive to the action of ethanol (where dilute concentrations of ethanol cause a perturbation equal to a large change in temperature) are those that exhibit a small thermal entropy change and a large difference in solute partitioning between membrane structures. Our model predicts that ethanol does not act on a single membrane structure, but on both structures in an equilibrium. The thermodynamic framework is applied to the action of ethanol on cooperative equilibria in a dipalmitoyl lecithin model membrane. Ethanol-induced perturbations are monitored by electron paramagnetic resonance (EPR) using the spin label, Tempo. The equilibrium between the gel and ripple-structures (L beta'-->P beta', pretransition) exhibits a small change in thermal entropy and, as predicted, is more sensitive to the action of ethanol than the equilibrium between the ripple and fluid bilayer-structures (P beta'-->L alpha, main transition) which exhibits a large thermal entropy change. The framework suggests that ethanol acts through entropy, as does temperature, thereby upsetting the natural thermal balance that maintains membrane architecture.

Nature of alcohol and anesthetic action on cooperative membrane equilibria.

A generalized, colligative thermodynamic framework is used to treat the action of solutes on cooperative membrane equilibria. Configurational entropy, the randomness imparted by solutes through the partitioning or mixing process, is implicated as the energetic driving force for the action of anesthetics on cooperative membrane equilibria. The equilibria predicted to be most sensitive to solute action--in which the dilute solute causes a perturbation equivalent to a large change in temperature--are (1) low-enthalpy processes that coincide with (2) large partitioning differences between states. The model stresses that solutes do not act at a single site, but on both states in an equilibrium, and that the perturbation is determined by the difference in entropy. Evidence for the thermodynamic framework is obtained from the partitioning behavior of the general anesthetic 1-hexanol into a model lecithin (DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine) membrane as a function of temperature and alcohol concentration. The low-enthalpy equilibrium between the gel (L beta') and ripple states (P beta') (pretransition) is more sensitive to 1-hexanol than the high-enthalpy equilibrium between the ripple (P beta') and fluid bilayer states (L alpha) (main transition). The perturbations of both equilibria are accurately described by the colligative thermodynamic framework. The results suggest that alcohols and anesthetics act through entropy to upset the natural thermal balance that maintains native membrane architecture.

Magnetic resonance chemical shift imaging and spectroscopy of atherosclerotic plaque.

An in vivo magnetic resonance imaging (MRI) technique for identification and characterization of atherosclerotic plaque was assessed in animal and human models. Atherosclerosis was induced in the abdominal aorta of four rabbits by a combination of balloon denudation and a high cholesterol diet. In vivo conventional spin-echo and fat/water suppressed images of the rabbit aortae were obtained at 1.5 T. Chemical shift imaging (CSI) was achieved using a hybridization of selective excitation and modified Dixon techniques. These techniques were then used to obtain images of atherosclerotic lesions in the carotid arteries of four patients prior to endarterectomy. The MRI results were corroborated by histologic and high-resolution proton MR spectroscopic (8.5 T) analysis of rabbit aorta, human carotid endarterectomy, and six additional human superficial femoral and iliac atherectomy specimens. All animal and human lesions were classified as either fatty streaks or fibrotic plaque. When compared to conventional spin-echo images, fat suppression by CSI substantially improved the measured contrast-to-noise ratio between plaque and vessel lumen, and enhanced its discrimination from periadventitial fat. In contrast, water suppression eliminated visualization of plaque due to the negligible amount of isotropic (liquid-like) signal from the immobilized lesion lipids. Magnetic resonance spectroscopy corroborated the CSI results by demonstrating broad, ill-defined fat resonances characteristic of nonmobile lipids in both human and rabbit atherosclerotic lesions. These findings indicate that in vivo MRI of plaque is technically feasible and can be markedly improved using chemical shift imaging.

The effect of chronic ethanol consumption on the fatty acid composition of phosphatidylinositol in rat liver microsomes as determined by gas chromatography and 1H-NMR.

Cell membranes and vesicles composed of extracted phospholipids isolated from rats chronically-fed ethanol develop a resistance to disordering by ethanol in vitro (membrane tolerance) and a decreased partitioning of ethanol into the membranes. The anionic lipid phosphatidylinositol (PtdIns) is the only microsomal phospholipid from the ethanol-fed rats that confers tolerance to vesicles of microsomal phospholipids from control rats in a paradigm where phospholipid classes are sequentially swapped. To investigate the molecular basis of this adaptation, the fatty acid content of microsomal PtdIns extracted from the livers of rats chronically fed ethanol for 5 weeks and their calorically-matched controls was analyzed by gas-liquid chromatography (GLC) and 1H-NMR spectroscopy. Chronic ethanol consumption caused an 8.4% decrease in arachidonic acid [20:4(n - 6)], a 20.0% increase in oleic acid [18: 1(n - 9)] and a 47.1% increase in the quantitatively minor fatty acid [20:3(n - 6)]. 1H-NMR was used to quantitatively assay compositional changes in the delta 5 olefinic moiety of the acyl chains in PtdIns, an approach that should be broadly applicable to other lipid systems. After chronic ethanol feeding PtdIns had decreased delta 5 unsaturates (-7.9% NMR, -8.2% GLC) and a corresponding increase in delta 5 saturates (+5.4% NMR, +5.3% GLC). In the other phospholipids, chronic ethanol feeding caused alterations in the fatty acid compositions specific for each phospholipid. PtdIns was the only microsomal phospholipid that exhibited a significant decrease in both the polyunsaturate pool and the ratio of the total olefinic content to the saturated fatty acid content. The major adaptive response in rat liver microsomal PtdIns to chronic ethanol administration involves a decrease in arachidonic acid [20:4 (n - 6)], which is partly compensated for by increases in oleic acid [18:1(n - 9)] and eicosatrienoic acid [20:3 (n - 6)], resulting in a depressed unsaturation and polyunsaturation index. The decreased unsaturation at the delta 5 position may have special functional relevance, due to the proximity of this position to the membrane surface, where ethanol is believed to reside. Whether these acyl changes are merely coincident with, or causative of, membrane tolerance requires further elucidation.