Lipid phase states influence glycophorin reconstitution.

Reconstitution of glycophorin into dimyristoyl phosphatidylcholine and sphingomyelin vesicles was sub-maximal below the phase transition temperatures of these lipids. Reconstitution of glycophorin into diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine liposomes was maximal within a range of temperatures below the phase transition temperatures of dimyristoyl phosphatidylcholine and sphingomyelin but above the phase transition temperatures of diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine. These findings indicate a greater tendency for reconstitution of glycophorin into fluid as opposed to solid lipid phases.

Trifluoperazine inhibits Sendai virus-induced hemolysis.

Sendai virus-induced hemolysis, a manifestation of virus-red cell fusion, is inhibited by exposure of the virus to 50 microM and higher concentrations of trifluoperazine. Trifluoperazine does not disrupt the virus, since trifluoperazine-treated virus with no hemolytic activity sediments slightly faster than untreated virus on sucrose density gradients and contains viral proteins in proportions characteristic of untreated virus. Trifluoperazine affects the fusion protein to a greater extent than the hemagglutinin, since trifluoperazine-treated virus with no hemolytic activity is as active or nearly as active in agglutinating red cells. The partition coefficient of trifluoperazine between the virus membrane and buffer is lower at 4 degrees C than, but the same at 37 degrees C, as that between the red cell membrane and buffer. Nevertheless, virus-independent red cell lysis and inactivation of virus-mediated hemolysis occur when the red cell and viral membranes, respectively, contain similar concentrations of trifluoperazine. Furthermore, 13-28% more trifluoperazine is necessary to achieve either effect at 4 degrees C or at 25 degrees C than at 37 degrees C. Changes in the surface activity of trifluoperazine do not explain these results, insofar as the critical micellar concentration of (0.75 mM) and maximal reduction in surface tension by (40 dyn/cm) trifluoperazine are the same at 25 degrees C and 37 degrees C. The fluorescence of viral tryptophan decreases by approx. 25% when viral hemolysis is inactivated by trifluoperazine, by trypsin treatment or by heating at 100 degrees C for 5 min.

Lipid mixing during freeze-thawing of liposomal membranes as monitored by fluorescence energy transfer.

A new pair of fluorescence-energy-transferring probes, dansylphosphatidylethanolamine and dioctadecylindocarbocyanine, were incorporated separately into phospholipid vesicles to monitor intervesicle lipid mixing under various conditions. The transfer efficiencies of mixtures of sonicated vesicles labeled with 2 wt% donor dansylphosphatidylethanolamine (DnsPE) or with 1 wt% acceptor dioctadecylindocarbocyanine (DiI-C18) were negligible, but increased to about 25% after the vesicles had been frozen in a solid CO2/ethanol bath, thawed and diluted. The freeze-thaw-induced mixing of lipids between vesicles, signified by energy transfer, was dependent on lipid concentration and was promoted by 0.5-1.5 M KCl, 0.5 M potassium trichloroacetate and 5 mM sodium acetate (pH 4) and inhibited by 0.5 M LiCl, 0.5 M glycerol, 0.5 M sucrose, 0.15 M KCl and 0.15-1.5 M NaCl. These results support and complement previously reported measurements of the trapped volumes, turbidities and population size distributions of similarly treated liposomes. Comparison of the responses of paucilamellar vesicles with those of multilamellar vesicles suggests that lipid mixing during freeze-thawing can occur either during interaction of the outermost bilayers of vesicles or during interaction of all bilayers, possibly as a result of breakdown and reformation of bilayer structure.

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles.

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Kienböck's disease: the role of silicone replacement arthroplasty.

A review of thirty-eight patients with Kienböck's disease indicated that both delay in diagnosis and prolonged treatment by plaster immobilization led to progressive collapse of the lunate and an unsatisfactory result in most instances. Four clinical and roentgenographic stages were identified, and the results of silicone replacement arthroplasty were correlated with the preoperative stage after an average follow-up of twenty-seven months. The results were satisfactory in fourteen of twenty wrists in which the procedure was performed before collapse of the lunate had occurred. The importance of early diagnosis and early surgical treatment of this disorder was emphasized by the findings in this study.

Personal accounts of satisfying and unsatisfying nursing experiences as a needs assessment strategy.

This article describes the assessment process by which learning needs of a select group of nurses in a forensic psychiatric setting were identified and used for continuing education (CE) program planning. To determine whether a gap existed between what the nurses currently knew and what they needed to know in order to have a sense of accomplishment, their personal accounts of satisfying and unsatisfying nursing experiences were analyzed. The factors that influenced these experiences were categorized according to a theory of basic human needs, and learning needs were identified from those categories. Implications for the planning of content and methodology of CE programs for the study group were then developed.

Optoelectronic switch matrix as a look-up table for residue arithmetic.

The use of optoelectronic matrix switches to perform look-up table functions in residue arithmetic processors is proposed. In this application, switchable detector arrays give the advantage of a greatly reduced requirement for optical sources by comparison with previous optoelectronic residue processors.

Phosphatidylserine vesicle lysis by Sendai virus at low pH is not due to virus-vesicle fusion.

As a model of the fusion of Sendai virus with red cells, the interaction of the virus with phosphatidylserine (PS) vesicles at pH 5 was quantitated by the release of a trapped marker from target vesicles and by mixing of lipids of the virus and the vesicles. Release of the marker was measured as dequenching of calcein trapped at a self-quenched concentration and lipid mixing was measured as a decrease in energy transfer between fluorescent phospholipid analogs in the target membrane. At comparable virus:vesicle ratios both calcein release and lipid mixing were maximal at pH 5 and significantly reduced after trypsin, but not chymotrypsin, treatment. In contrast, these two effects differed in their PS dependence, time course, and temperature dependence, indicating that calcein release is not a consequence of the fusion of a permeable virus membrane with an impermeable target membrane. Vesicles composed of 25 to 100% PS released similar amounts of calcein, whereas fusion increased linearly as a function of PS content of the target vesicles. The half-time was 15 s for calcein release but 1.5 min for fusion. The temperature coefficient of fusion was at least three times greater than that of calcein release. These results indicate that calcein release at pH 5 may signify an interaction of the virus with PS target membranes which precedes but does not necessarily culminate in fusion, given too low a temperature or an inappropriate target membrane composition.

Fully orthogonal optical-code multiplex for broadcasting.

A novel method of optical-code multiplex transmission from a central location is proposed. It has the advantages that the receivers can be configured to any channel quickly, the channels have in principle zero cross talk, and the bandwidth-expansion factors are less than for other optical-code-division multiple access arrangements. The proposed method is based on arrays of optoelectronic switching detectors that are at present under development for broadband matrix switching.

Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose.

To determine whether polyethylene glycol (PEG) causes growth of liposomes by affecting them directly or indirectly, vesicles composed of phosphatidylcholine were exposed to increasing concentrations of Mr 15 000-20 000 PEG or Mr 40 000 dextran either by direct mixing or across a dialysis membrane. After incubation at room temperature and dilution below at least 5% (w/w) polymer, the vesicles were monitored for fluorescence energy transfer and for absorbance at 400 nm. PEG induced the same levels of dequenching or lipid mixing and increased turbidity, regardless of whether the vesicles had been mixed directly with or dialyzed against PEG. These changes occurred within 5-15 min of polymer application. It is concluded that the increased lipid mixing and/or increased turbidity, indicating vesicle growth, resulted from an indirect effect of PEG on the vesicles--most likely dehydration. Dextran, in contrast to PEG, induced less dequenching and/or less turbidity increase when vesicles were directly mixed with, as opposed to dialyzed against, dextran. Although dextran not in contact with vesicles and with osmotic activity comparable to PEG was able to cause a degree of membrane fusion similar to that of PEG, therefore, the dehydrating effect of dextran could be mitigated if it were allowed to interact with vesicles. In further support of membrane dehydration as a precursor to membrane fusion, lipid mixing among sonicated and sonicated, frozen-thawed vesicles dialyzed against sucrose increased as a function of sucrose concentration. Vesicle morphology generally determined the maximal degree of membrane fusion inducible by the polymers.(ABSTRACT TRUNCATED AT 250 WORDS)

High gain optical detection with GaAs field effect transistors.

The photoresponse of GaAs field effect transistor (FET) optical detectors to optical intensity modulation signals of moderate frequency is investigated. High ac responsivity is available at frequencies of interest in optical communications: more than 6 A/W can be obtained up to 100 MHz, with a noise equivalent power of <10(-12) W/ radicalHz. The slope of the frequency response is dependent on the otpical bias power, an effect that can be attributed to traps. The GaAs FET optical detector complements the performance of available P-I-N and avalanche photodiode photodetectors for communications by providing high photoresponse at low bias voltages.

Frequency domain optical reflectometer.

A correlation reflectometer operating in the frequency domain is described. It is shown that such an instrument is well adapted to detecting weak discrete reflections. The detection of end reflections in a 2.2-km length of fiber whose end is index matched is demonstrated. A round-trip range of over 70 dB is obtained with a 1-mW optical source power. The method shows promise for characterizing the reflective properties of optical fiber elements, such as tapers, microbends, and splices, and may be useful in fault location.

Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes.

Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol membranes. The calculated R0 for 50% efficiency of energy transfer from excited state probe to monomer was 55-58 A and to dimer was 27 A. Since lateral diffusion of C18-Rh is probably too slow to permit collisional quenching during the lifetime of the probe, even if C18-Rh were concentrated in a separate phase, C18-Rh self-quenching appears to be due mainly to energy transfer without emission to nonfluorescent dimers.

Energy transfer measurements of fusion between Sendai virus and vesicles corrected for decreased absorption of acceptor probe.

The fusion of Sendai virus at pH 4-7 with artificial lipid vesicles composed of phosphatidylserine or phosphatidylcholine was quantified by measuring fluorescence energy transfer from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine to N-(lissamine-rhodamine-B-sulfonyl)-phosphatidylethanolamine in the target membranes. About 60% of the phosphatidylserine vesicles and virus appeared to fuse at pH 4 and about 100% at pH 5. Fusion was much less under all other conditions. The apparent fusion at pH 4, however, was due to a decrease in absorption of the acceptor probe, instead of dilution of acceptor as a result of fusion of labeled vesicles with unlabeled virus. After correction for this fusion-independent effect of Sendai virus, the extent of fusion was only 4-20% at pH 4 but still 80-100% at pH 5. These findings paralleled the loss of hemagglutinating and hemolytic activities of the virus induced by incubation at pH 4 but not at pH 5. Vesicle-virus hybrids were observed with the electron microscope after incubation at pH 5 but not at pH 7. The assay of membrane fusion by fluorescence energy transfer can be misleading unless correction is made for changes in energy transfer due to fusion-independent effects.

Temperature and ionic effects on the interaction of erythroid spectrin with phosphatidylserine membranes.

Specific binding of human erythroid spectrin to large, unilamellar vesicles of bovine brain phosphatidylserine, made by an extrusion technique (LUVETs), has been measured and characterized by a new gel filtration assay. Vesicle-bound spectrin was separated from free spectrin by Sepharose CL-2B chromatography and detected by its intrinsic (tryptophan) or extrinsic (carboxyfluorescein) fluorescence. That the bound spectrin was not an aberrant, adhesive form was shown by the ability of a portion of free spectrin, which had not bound to PS LUVETs during a previous incubation, to bind during a subsequent incubation. Spectrin binding reached a plateau by 30 min of incubation at room temperature and at 37 degrees C. Binding increased from a low level below 31 degrees C to about twice as much as 37 degrees C and to 4-7 times as much between 40 and 43 degrees C. Similar results were obtained with LUVETs composed of DOPS but not PC. Triton treatment of PS LUVETs and spectrin after incubation of spectrin and vesicles at 40 and 43 degrees C but prior to chromatography on Sepharose CL-2B eliminated the bound spectrin peak, which thus did not consist of large aggregates of covalently associated spectrin. Binding isotherms fit by nonlinear regression gave an apparent Kd of 0.31 microM and an apparent maximum spectrin binding of 33 nM/mM PS at 25 degrees C, an apparent Kd of 0.35 microM and an apparent maximum spectrin binding of 40 nM/mM PS at 31 degrees C, and an apparent Kd of 3.4 microM and an apparent maximum spectrin binding of 113 nM/0.1 mM PS at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Swept wavelength reflectometer for integrated-opticmeasurements.

The experimental investigation of a high-resolution optical reflectometry technique based on the thermally induced sweep of sample refractive index is described. Reflections from surface scratches and the end surfaces of a Ti:LiNbO(3) waveguide were detected with a resolution of <0.2 mm, which is sufficient for the characterization of reflections in integrated-optic devices. Improvement in the dynamic range of the measurement is required for the detection of well-fabricated waveguide features such as bends and Y junctions. Several possibilities exist for making this improvement.