E-genetics: exploring the acceptability and feasibility of using technology in cancer genetics services.

The use of information and communication technologies (ICTs) in the delivery of cancer genetics services could improve equality of access in rural areas and help meet the increasing demand for specialist genetics services. An online patient survey and focus groups with patients and staff from the Cancer Genetics Service for Wales (CGSW) were used to explore the acceptability and feasibility of utilizing ICTs within genetics services, which we have termed e-genetics. A total of 225 patients completed the online survey. Many aspects of e-genetics proposed in the survey were highly acceptable to patients, including an electronic version of the family history questionnaire, an email facility for cancer genetic queries, and a computerized decision-aid. Participants in the focus groups emphasized the importance of patient choice when developing new models of service delivery. For example, the use of genetic counselling via telemedicine was not considered to be preferable to face-to-face clinic appointments but could benefit those unable to travel. This article highlights the fact that e-genetics initiatives may not be appropriate for all cancer genetics service users. However, user-friendly developments that can be easily implemented and attend to individual needs could improve efficiency and cost-effectiveness, whilst providing high-quality services to remote areas.

Providing evidence-based answers to clinical questions. A pilot information service for general practitioners.

OBJECTIVE: To pilot a clinical information service for general practitioners. METHODS: A representative sample of 31 GPs was invited to submit clinical questions to a local academic department of general practice. Their views on the service and the usefulness of the information were obtained by telephone interview. RESULTS: Over one month, nine GPs (29% of the sample, 45% of those stating an interest), submitted 20 enquiries comprising 45 discrete clinical questions. The median time to search for evidence, appraise it and write answers to each enquiry was 2.5 hours (range, 1.0-7.4 hours). The median interval between receipt of questions and dispatch of answers was 3 days (range, 1-12 days). CONCLUSIONS: The GPs found the answers useful in clinical decision making; in four out of 20 cases patient management was altered.

Permeability studies on liposomes formed from polymerizable diacetylenic phospholipids and their potential applications as drug delivery systems.

We have investigated the permeability and entrapment characteristics of liposomes formed from a group of polymerizable phospholipids, containing diacetylenic groups in one or both of their acyl chains. Permeability was assessed by the release of an entrapped dye, 6-carboxyfluorescein. Diacetylenic phosphatidylcholine (PC) liposomes were found to exhibit a wide range of permeability properties, depending on: the nature of the diacetylenic lipid, i.e. mixed-chain (mc) or identical-chain (id), the extent of polymerisation, vesicle size, and cholesterol content. Ultraviolet-initiated polymerisation affected a significant decrease in the permeability of C25idPC liposomes. The increase in permeability of liposomes formed from four other diacetylenic lipids (C25mcPC, C23idPC, C23mcPC and C20idPC) after polymerisation was attributed to disturbances in the packing of lipid molecules, and/or the limited ability of small unilamellar vesicles to accommodate long polymers. The C20idPC lipid is atypical, forming irregular monomeric and polymeric vesicles. The permeability of C25idPC liposomes was also assessed by the release of [3H]inulin. C25idPC liposomes exhibited low permeabilities to [3H]inulin in their monomeric and polymeric states. Incubation of C25idPC liposomes in human plasma caused a substantial increase in the permeability of monomeric vesicles to both carboxyfluorescein and [3H]inulin. The permeability of polymerised C25idPC liposomes, however, was unaffected in the presence of plasma, with vesicles retaining most of their entrapped [3H]inulin after 50 h. These findings demonstrate that polymeric C25idPC liposomes exhibit high resistance to the destructive actions of plasma components, such as high-density lipoproteins (HDLs). Polymeric C25idPC liposomes may have an application in drug delivery systems.

Biomembranes as models for polymer surfaces. IV. ESCA analyses of a phosphorylcholine surface covalently bound to hydroxylated substrates.

Using a simple chemical process, phosphorylcholine has been deposited covalently on the surface of a variety of hydroxylated polymers as a stable, monomolecular coating. Our goal was to obtain new biomaterials which, due to the chemical similarity of the modified interfaces to the phospholipid head groups present on the extracellular surfaces of blood cell membranes, should exhibit enhanced haemo- and biocompatibility. Our previous analyses by chemical and spectrophotometric methods indicated that sufficient quantities of phosphorylcholine were deposited on glass and silica surfaces to result in appreciable modification of their interfacial properties. In the present study, we have examined a series of modified hydroxylated substrates by ESCA and demonstrate specific chemical modifications on the molecular surfaces of polymeric substrates.

Biomembranes as models for polymer surfaces. II. The syntheses of reactive species for covalent coupling of phosphorylcholine to polymer surfaces.

We have proposed previously that the biocompatibility of biomaterial surfaces might be improved by mimicking the phospholipid components which are present on the external faces of cellular plasma membranes. This approach may have particular relevance to the generation of haemocompatible surfaces, since the distributional asymmetry of phospholipids in the plasma membranes of blood cells is critical to the maintenance of haemostasis. The simplest feature common to the external faces of essentially all the mammalian plasma membranes investigated so far is the high content of the electrically neutral, but zwitterionic, phosphorylcholine. The present communication describes the syntheses of a group of novel reactive species capable of covalently linking phosphorylcholine to a variety of polymer surfaces.

Biomembranes as models for polymer surfaces. III. Characterization of a phosphorylcholine surface covalently bound to glass.

A surface layer of phosphorylcholine has been chemically linked with the surface hydroxyl groups present on glass and silica by reaction with mono- and bifunctional reagents. Evidence for the structural integrity of the deposited group was provided by the equimolar association of phosphorus and choline with the reacted surfaces. Modified glass surfaces yielded contact angles which are consistent with those found previously for other models of biological membranes. Covalent modification of the treated surfaces was demonstrated by i.r. spectroscopy via the removal of surface hydroxyl groups. The modified surfaces were thermostable at temperatures up to 375 degrees C for extended periods. The relevance of these results to the generation of new biomaterials is discussed.

Polymerized liposomes as stable oxygen-carriers.

We have produced a surrogate erythrocyte ('hemosomes') by encapsulating human hemoglobin in polymerized vesicles composed of diacetylenic phospholipids plus or minus cholesterol. Hemoglobin (in the presence or absence of allosteric effectors) was encapsulated by a freeze-thaw method in large, unilamellar vesicles composed of monomeric lipids. Entrapment was demonstrated by molecular-sieve chromatography. Brief irradiation with ultraviolet light produced polymeric hemosomes with polymerization kinetics and conversions similar to liposomes in the absence of protein. Photo-induced oxidation of the heme was eliminated or severely limited by a combination of prior ligation with CO and the maintenance of high intravesicular hemoglobin concentrations (5-10 mM internal hemoglobin). The inclusion of allosteric effectors within polymerized hemosomes facilitated near-quantitative conversion to the oxy-HbA form. Gas permeability of monomeric and polymeric hemosomes was demonstrated by spectroscopic methods. Reversible spectral shifts, corresponding to oxygenation-deoxygenation, were obtained after brief evacuation and exposure to oxygen or nitrogen. The gas permeability of polymerized hemosomes appears sufficient for the vesicles to act as oxygen carriers in vivo, a notion that is strengthened by their apparent hemocompatibility.

Second-derivative infrared spectroscopic studies of the secondary structures of bacteriorhodopsin and Ca2+-ATPase.

The resolution of minor amide components in the infrared spectra of membrane proteins has, in the past, been limited by the small differences in frequency compared to the large half-widths of the bands that are assigned to different secondary conformations. Here, second-derivative calculations are used to resolve the relatively weak bands that are associated with the beta-sheet conformation and the vibrations of some amino acid side chains in the infrared spectra of bacteriorhodopsin and Ca2+-activated adenosine-5'-triphosphatase (Ca2+-ATPase). The spectra presented indicate that bacteriorhodopsin in the purple membrane contains an appreciable amount of beta structure in addition to the predominant alpha II-helical structure. Both sarcoplasmic reticulum and purified Ca2+-ATPase in native lipids contain alpha-helical and random coil conformations together with a small amount of beta structure. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Ca2+-ATPase adopts a secondary conformation similar to that in the sarcoplasmic reticulum, and this structure is unaffected by the phospholipid phase transition. A shift to a predominantly random coil conformation is associated with solubilization of both bacteriorhodopsin and Ca2+-ATPase in 20% Triton X-100. Second-derivative analysis of the carbonyl stretching vibrations of DMPC bilayers indicates that below the phase-transition temperature (Tm) both bacteriorhodopsin and Ca2+-ATPase perturb the interface region such that the sn-2 carbonyls adopt a conformation similar to the sn-1 carbonyls. Above Tm, these integral proteins have no effect on the static order of the interface region, and the conformational inequivalence of the sn-1 and sn-2 carbonyls is similar to that found in a pure lipid bilayer.

Polymeric phospholipids as new biomaterials.

Phospholipid polymers form a new class of biomaterials with many potential applications in medicine and research. The development of these compounds is based upon the mimicry of cell surfaces and reflects our current understanding of the properties of membrane lipids. Physicochemical characterization of the monomeric, diacetylenic phospholipids illustrates the similarities to naturally occurring lipids, similarities that are confirmed by the capacity to enrich the membranes of A. laidlawii to the level of 90% diacetylenic lipid. Polymerization of diacetylenic phospholipids is easily attained by irradiation and produces a stable, crystalline array. The ability to link membrane lipids covalently permits the isothermal restriction in their motion, and is useful in basic studies of biomembranes. The thromboresistance of polymeric phosphatidylcholines in vitro may be a consequence of the inability of phosphatidylcholines to participate in coagulation. The restricted lateral diffusion of proteins along a polymeric lattice will also inhibit the formation of coagulation complexes. Existing polymers may be altered by a coating of polymeric lipid obtained by the Langmuir-Blodgett method. Polymerized vesicles display significant reductions in permeability and aggregation. Entrapment of soluble materials and reconstitution of membrane proteins may be exploited in controlled and site-directed drug delivery. Polymerization of cells in situ produces "cellular capsules" with entrapped membrane and cellular components. Polymeric hemosomes are capable of gas transport and may function as red cell surrogates. The hybrid qualities of biomembranes (polar surfaces, nonthrombogenic, low antigenic potential, and low permeability) and synthetic polymers (chemical and physical stability) suggest that polymeric phosphatidylcholines may serve as models for biomaterials design.

Biomembrane surfaces as models for polymer design: the potential for haemocompatibility.

A major restriction in the application of polymeric biomaterials is the propensity of their surfaces to support thrombosis. Theoretical approaches to the design of thromboresistant polymers have been inadequate because of the complexity of surface thrombosis. We have developed a new, practical approach to this problem--the design of polymers which mimic the thromboresistant surfaces of blood cell membranes. Haemostatic processes are mediated by reactions which occur at membrane-plasma interfaces. The extra-cellular surfaces of the plasma membranes of red blood cells and quiescent platelets are thromboresistant; in contrast, their cytoplasmic surfaces are thrombogenic. The simplest common feature among the blood-compatible cellular and model membranes is the high content of the electrically neutral phospholipids which contain the phosphorylcholine head group. We have developed model systems of biological membranes which utilize polymerizable phosphatidylcholines and which mimic nonreactive cell surfaces. Polymeri phospholipids represent a new class of hybrid biomaterials with characteristics both of biomembranes (polar surfaces, nonthrombogenic, low antigenic potential and low permeability) and of synthetic polymers (chemical and physical stability).

Cholesterol levels and plasma membrane fluidity in 3T3 and SV101-3T3 cells.

Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18: 739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101-3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101-3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101-3T3 cells.