Interfacial free energies of intact and reconstituted erythrocyte surfaces. Implications for biological adhesion.

Model cell surfaces consisting of phospholipids or phospholipids and the erythrocyte membrane glycoprotein glycophorin have been formed at an oil/water interface. Interfacial free energies have been estimated from surface wetting by both hydrophobic and hydrophilic test droplets on both the model surfaces and on intact erythrocytes. The use of a dense fluorocarbon oil to form the oil/water interface facilitates analysis by minimising surface deformation by the test drop. Hydrophobic test droplets (polar hydrocarbon oils) show increasing contact angles (decreasing wetting) with increasing hydrophilicity (decreasing interfacial free energy) of the model interface. Hydrophilic test droplets (phase separated aqueous polymer systems) show the opposite behaviour spreading more as the interfacial free energy is decreased. Both systems give similar estimates of the interfacial free energy. Glycophorin reproduces the wetting properties of intact cell surfaces by reducing the lipid-water interfacial free energy from 5.10(-3) J . m-2. From molecular considerations it is concluded that 'cell surface free energy' is an ambiguous term; its magnitude depends on the location of the interface in question. Thus, in a thermodynamic analysis of interactions at biosurfaces (such as cellular adhesion, chemotaxis or membrane fusion), the interfacial free energies may vary by more than three orders of magnitude depending on the location of the particular interface.

A synthetic emulsion reproduces the functional properties of pulmonary surfactant.

An effective substitute for pulmonary surfactant must be able to reduce water-vapour surface tensions to under 1 mN/m and it must spread rapidly and spontaneously at the air interface from the aqueous phase. Pure dipalmitoylphosphatidylcholine meets the former requirement, but not the latter. A synthetic surfactant is described which meets both of these criteria. The surfactant is prepared as a DPPC monolayer stabilizing an aqueous emulsion of an inert fluorocarbon oil; it spreads rapidly at the air/liquid interface, lowers the surface tension to under 1 mN/m during compression at the air/liquid interface and restores normal pressure-volume characteristics to surfactant-depleted lungs.

Determination of cell/medium interfacial tensions from contact angles in aqueous polymer systems.

The contact angles on cell layers of a series of polymeric droplets from aqueous two-phase systems of dextran and poly(ethylene glycol) have been used to determine the critical or limiting interfacial tension for spreading on the cell layers. Test droplets of the denser dextran-rich phase were formed in the lighter poly(ethylene glycol)-rich phase. The interfacial tensions, gamma, between the phases were determined with the pendant drop method, and a linear relationship was found between gamma-1/2 and the cosine of the angle the droplets made with the cell layers (Good-Girifalco plot). We were thus able to determine the limiting or critical interfacial tension, gamma c, for spreading on the cell layers. The value of gamma c is a measure of the interfacial energy of the cell/bathing medium interface. Values of gamma c obtained by this method include the following: 0.65 and 0.84 microN . m-1 for human erythrocytes and neutrophils, respectively; 0.93 microN . m-1 for porcine pulmonary macrophages; 0.75--3.60 microM . m-1 for various transformed murine lymphoid cell lines, and 2.53 microN . m-1 for Balb/c murine spleen lymphocytes. Exposure to various agents has differing effects on gamma c. Concanavalin A reduces gamma c, and bacterial lipopolysaccharide increases gamma c of murine spleen lymphocytes. The calcium ionophore, A23187, increases gamma c of both porcine pulmonary macrophages and murine spleen lymphocytes. This new method provides a quantitative approach to the cell surface energy and hydrophobicity which are thought to play an important role in membrane-mediated phenomena and in cell adhesion.

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells.

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.

Effects of elevated pressures of inert gases on cytosolic free Ca2+ of human platelets stimulated with ADP.

Platelets were exposed to elevated pressures of helium (He), nitrogen (N2), and He/xenon (He/Xe, 85/15), and stimulated with ADP (5 microM). He to 36 atmospheres absolute (ATA) inhibited the ADP-stimulated increase in [Ca2+]i, measured by Fura-2/AM, with most of the effect occurring by 9 ATA. N2 caused a similar but initially greater effect and it was bimodal, with inhibition being less at 36 ATA than at 18 ATA. N2 also significantly depressed basal levels at 18 and 36 ATA. In the He/Xe mixture, the effect on ADP-stimulated [Ca2+]i was no different from He alone at the same pressures, but basal levels were significantly depressed. In a calcium-free medium both He and N2 moderately depressed the already-reduced response to ADP stimulation but only N2 significantly depressed basal levels at all pressures. These results indicate that raised pressures of inert narcotic gases, as well as pressure per se (represented by He), can affect cell [Ca2+]i sufficiently to have physiological consequences. This, together with previous findings in this laboratory, may have implications for some of the neurological problems associated with deep diving.

Effects of elevated pressures of inert gases on cytosolic free Ca2+ of cultured human neuroblastoma cells stimulated with carbachol: relevance to high pressure neurological syndrome.

Suspended cells of the human neuroblastoma line SK-N-SH were exposed to elevated pressures of non-narcotic helium (He) and the narcotic gases nitrogen (N2), and argon (Ar) and stimulated with carbachol. He, 18 and 36 atmospheres absolute (ATA), equivalent to 544 and 1120 feet of seawater, potentiated the increase in [Ca2+]i induced by carbachol, as measured by Fura-2. Carbachol-stimulated increases in [Ca2+]i were not significantly altered from values in 1 ATA air by either N2 or Ar at the same pressures. The response to carbachol of cells exposed to 36 ATA of He and slowly decompressed to 1 ATA was indistinguishable from that of cells never exposed to pressure. Thus this pressure-potentiated increase in [Ca2+]i is compatible with excitation, is reversible and is not elicited by narcotic gases. It was observed, moreover, at pressures encountered by commercial deep-sea divers. The High Pressure Neurological Syndrome (HPNS) encountered by divers breathing He/O2 mixtures at high pressures, and its known antagonism by N2, may be due in part to effects on neuronal [Ca2+]i levels since an increase in these would most likely result in an excitatory response.

Interfacial tensions in healthy and atherosclerotic rabbit aortae. Higher values and lesion surfaces.

Interfacial tensions at the saline/arterial wall interface were determined by measuring contact angles between various test fluid droplets and the walls of rabbit aortae immersed in physiological saline. These contact angles and the interfacial tensions of the test fluid/bathing fluid interface (measured by the Du Noüy ring method) were converted to saline/arterial wall interfacial tensions by applying Neumann's equation of state. Four diseased animals, fed an atherogenic diet for 6-8 weeks and 6 controls formed the experimental group. A significantly higher interfacial tension (P < 0.001), was determined for lesion surfaces in atherosclerotic arteries (0.36 +/- 0.08 (SEM) mM . M-1, n = 13) compared to both the surrounding undisturbed regions (0.035 +/- 0.01 mN . m-1, n = 14) and the intact surface of control vessels (0.060 +/- mN . m-1, n = 48). This increase may reflect a change in the strength of hydrophilic interactions associated with the lesion surface in atherogenesis.

The medical usefulness of Stat tests.

Although Stat tests are one of the most important features of clinical laboratory performance, the indexed literature is devoid of significant discussion of the subject. The authors, undertook a study by querying hospital laboratories about their current practices and received 38 useable answers dealing with the types of tests done on an urgent basis, the turn-around time for reports, the limitations on ordering, the use of special Stat laboratories and instruments, and decisions regarding priorities.

Activated neutrophils impair rabbit heart recovery after hypothermic global ischemia.

Cardiopulmonary bypass is known to cause neutrophil activation, and activated neutrophils appear to be of importance in myocardial reperfusion injury. This study examined the effect of a preischemic infusion of activated neutrophils on the recovery of myocardial function after 40 minutes of hypothermic global ischemia. Studies were carried out in three groups of Langendorff-perfused rabbit hearts: control, control (unactivated) neutrophil infusion, and phorbol myristate acetate-activated neutrophil infusion. The activated neutrophil group showed significant deterioration in function during the activated neutrophil infusion. All three groups demonstrated significant depression of function initially after reperfusion, but the two control groups subsequently recovered to baseline levels. The activated neutrophil group, however, showed a persistent significant depression in ventricular force, rate of ventricular tension development, and rate of ventricular relaxation as well as a significant increase in coronary vascular resistance. It is concluded that activated neutrophils depress myocardial function and contribute to impaired recovery of function after global hypothermic ischemia.

Activated neutrophils depress myocardial function in the perfused rabbit heart.

OBJECTIVE: To examine the effect of activated neutrophils on myocardial function in the absence of ischemic and/or reperfusion injury. DESIGN: Studies were carried out in five groups of Langendorff perfused rabbit hearts: control; nonactivated rabbit neutrophil infusion; phorbol myristate acetate-activated rabbit neutrophil infusion; nonactivated human neutrophil infusion; and phorbol myristate acetate-activated human neutrophil infusion. RESULTS: Both groups receiving activated neutrophils showed significant deterioration in ventricular force, rate of ventricular force development (dF/dt) and rate of ventricular relaxation (-dF/dt), and significant increases in coronary vascular resistance. Myocardial lipid peroxidation was assessed but there was no significant difference among groups. Myocardial prostacyclin production was significantly increased in hearts receiving the phorbol myristate acetate-activated human neutrophil infusion. CONCLUSION: It is concluded that myocardial perfusion with activated neutrophils results in depression of myocardial function and an increase in coronary vascular resistance even in the absence of ischemic/reperfusion injury.

Deoxyribonucleic acid hybridization method for the detection of Listeria in dairy products, seafoods, and meats: collaborative study.

The method is based on the hybridization of synthetic deoxyribonucleic acid probes to ribosomal ribonucleic acid sequences unique to Listeria. This method was compared to 2 culture methods: the U.S. Food and Drug Administration method for the detection of Listeria in dairy products and seafoods and the U.S. Department of Agriculture, Food Safety and Inspection Service method for Listeria in meats. Six food types with replicate samples containing various concentrations of Listeria were analyzed by the collaborating laboratories. Listeria was detected in 774 samples using the DNAH method and in 772 samples using a culture method. The DNAH and culture methods were in agreement for 668 samples containing Listeria and 306 samples without Listeria. The overall rate of agreement between methods was 82.3%. The method has been adopted first action by AOAC INTERNATIONAL.

Serum levels of immunoglobulins A, G and M in New Zealand and Tokelauan children.

Serum levels of immunoglobulins A, G and M are described in a group of New Zealand (NZ) children of European descent. These findings are used as reference standards for three groups of Tokelauan children, atoll residents, migrants in NZ, and NZ-born Tokelauan children. All three Tokelauan groups had high levels of IgA, IgG, and IgM. Breast feeding was diminished and morbidity was higher in the NZ-born group. The IgG level was negatively correlated with duration of breast feeding in this group. In the atoll group IgG was related to present illness but no other consistent relationships were found between present illness status and the immunoglobulin levels. It remains unclear whether the high Tokelauan immunoglobulin levels are genetically or environmentally determined.

Effects of high hydrostatic pressures on secondary structure of acetylcholinesterase with and without carbachol.

Ultra-high hydrostatic pressures (to 13 kbar) were applied to acetylcholinesterase (AChE) in the presence and absence of 1 mM carbachol (a muscarinic agonist) by means of a piston-and-cylinder system designed for use with Fourier transform infrared spectroscopy. At normal atmospheric pressure, carbachol decreased the number of intramolecular hydrogen bonds and the anti-parallel beta-sheet structure. In the absence of carbachol, pressure dramatically increased the number of intermolecular hydrogen bonds but decreased the alpha-helical, beta-sheet, and anti-parallel beta-sheet segments. In the presence of carbachol, pressure had the opposite effects, decreasing the number of intermolecular hydrogen bonds and increasing the alpha-helix: beta-sheet ratio. Thus in the absence of an attached ligand, the enzyme molecule was vulnerable to pressure-induced distortions that would most likely impair its function. These effects were observed in the absence of a lipid component, indicating that pure proteins are vulnerable to pressure-induced changes in configuration that could affect function.

Thermal anomalies in acetylcholine release: evidence for control of membrane fusion by interfacial water.

Thermal anomalies in the rate of acetylcholine release from electrically stimulated guinea-pig ileum have been demonstrated at 18 degrees C and 30 degrees C. Because similar anomalies occur in many aqueous systems where interfacial water plays a dominant role, it is concluded that the anomalies in acetylcholine secretion may indicate domination of the release process by interfacial water. Sharp breaks in the Arrhenius plots at the temperatures of the anomalies separate non-linear portions of positive gradient from a linear portion of negative gradient. The negative curve probably results from the rate of a competitive reaction (such as the rate of removal of calcium from the nerve terminal) while the positive portions may arise from cooperative H-bonding of water molecules. It is proposed that water-dominated phase transitions in membrane lipids may also mediate the effects of both temperature and calcium ions on neurotransmitter release in particular and membrane fusion in general.

Foulds' "general instability" and "psychopathy" 16PF scales and their relationship to psychiatric mood state.

Previous research examined "general instability" and "psychopathy" scales, derived from the 16PF, in terms of Foulds' criteria of content, group differentiation, change over time, and score distributions. When the external criterion of a newly validated measure of psychiatric mood state (the DSSI/sAD) was used, it was confirmed in both a patient and a normal group that the "general instability" scale is related significantly to symptomatology, while the "psychopathy" scale is relatively independent of present state.

The influence of electrolytes on the volume of non-metabolizing renal cortical cells.

1. The metabolism of rat renal cortex slices was inhibited by iodoacetate and anoxia, and swelling was prevented by the presence in the medium of 7.2 g polyethylene glycol 6000/100 ml. (referred to as PEG medium).2. Slices were incubated for up to 12 hr in PEG medium, and in PEG media containing 440 m-osmole/kg H(2)O of an electrolyte (LiCl, NaCl, KCl or RbCl), or a non-electrolyte (glucose).3. It was concluded that the slices in all media were at equilibrium with the medium after incubation for 8 hr.4. Slices in the medium containing glucose reached the same equilibrium water content as those in the PEG medium, but slices in all the electrolyte media had significantly lower equilibrium water contents, although these did not differ significantly from each other.5. It is suggested that the results demonstrate a non-specific effect of electrolytes on the swelling of non-metabolizing cells.

Extracellular space in some isolated tissues.

1. The spaces occupied by isotopically labelled inulin, polyethylene glycol, mol. wt. 4000 (PEG 4000), polyethylene glycol, mol. wt. 1000 (PEG 1000) and sucrose in metabolizing mammalian kidney and liver slices and in toad bladder epithelial cell preparations incubated in vitro have been examined.2. In slices of mammalian tissue, and in homogenized liver, it proved impossible to extract inulin completely from tissue which had been dried. However, inulin was recovered as completely from both dried and undried toad bladder epithelial cells scraped from hemibladders incubated in vitro.3. PEG 4000 occupied a space in all preparations similar to that from which inulin was extracted in dried tissue.4. PEG 1000 and sucrose entered cellular water in mammalian slices, but PEG 1000 occupied a similar space to inulin in toad bladder epithelial cell preparations.6. It is concluded that inulin enters cellular water in mammalian slices from which after drying of the slices it cannot be extracted. It thus rather fortuitously provides a measure of extracellular water under these conditions. In preparations of toad bladder epithelial cells inulin seems to be a satisfactory extracellular marker. PEG 4000, which did not appear to enter cellular water also allows a reasonable estimate of extracellular water. PEG 1000 is a suitable extracellular marker for toad bladder epithelial cell preparations but not for mammalian slices. Sucrose entered cellular water in both slices and toad bladder epithelial cells and is not a satisfactory extracellular marker in these tissues.

Effects of gaseous anesthetics and ultrashort and short-acting barbiturates on human blood platelet free cytosolic calcium: relevance to their effects on platelet aggregation.

The effects of elevated pressures (to 6 atmospheres absolute (ATA)) of nitrous oxide (N2O) and of xenon (Xe), and barbiturates on platelet free cytosolic calcium ([Ca2+]i) and platelet aggregation were studied. N2O inhibited the ADP-induced rise in [Ca2+]i whereas Xe had no effect. Neither affected basal levels. Pentobarbital and methohexital had little effect on basal or stimulated levels in the presence or "absence" of extracellular Ca2+; but both, at concentrations > 10(-4) M, inhibited platelet aggregation induced by adenosine diphosphate. Thiopental increased basal and stimulated [Ca2+]i when extracellular Ca2+ was present, but not when it was absent, and displayed a bimodal effect with low and high doses being more active than intermediate ones. It also potentiated aggregation. Methitural displayed similar, but nonsignificant, effects. These patterns held for all agents whether or not acetylsalicylic acid was present. Pentobarbital and methohexital inhibited phorbol myristate acetate aggregation in low extracellular calcium and no potentiation was seen with thiopental. In the absence of extracellular Ca2+, no potentiation was observed in stimulated platelets. Potentiation of aggregation previously reported for Xe does not involve increased Ca2+ uptake and did not occur in the absence of extracellular Ca2+. A common mechanism of action for these agents cannot be inferred from their effects on platelet aggregation or [Ca2+]i, as their pharmacological profiles differ markedly. It is evident that their inhibitory properties in this cell are not dependent on extracellular Ca2+, whereas the potentiation observed with pentobarbital, and formerly with Xe, is so dependent.

Deuterium oxide protects against hyperthermic cell death: evidence for role of interfacial water.

Tissue culture cells incubated at 43 degrees C show a dramatic decrease in cell survival compared with cells incubated at lower temperatures; the effect is termed hyperthermic cell death (HCD). Replacing water in the culture medium with deuterium oxide produces a dose-dependent reduction in HCD. On the basis of this and other evidence it is concluded that the mechanism of HCD involves a hydration-dependent phase transition in membrane lipids.