Are digital images good enough? A comparative study of conventional film-screen vs digital radiographs on printed images of total hip replacement.

The aim of this study was to evaluate the inter- and intra-observer variability and to find differences in diagnostic safety between digital and analog technique in diagnostic zones around hip prostheses. In 80 patients who had had a total hip replacement (THR) for more than 2 years, a conventional image and a digital image were taken. Gruen's model of seven distinct regions of interest was used for evaluations. Five experienced radiologists observed the seven regions and noted in a protocol the following distances: stem-cement; cement-bone; and stem-bone. All images were printed on hard copies and were read twice. Weighted kappa, kappa(w), analyses were used. The two most frequently loosening regions, stem-cement region 1 and cement-bone region 7, were closely analyzed. In region 1 the five observers had an agreement of 86.75-97.92% between analog and digital images in stem-cement, which is a varied kappa(w) 0.29-0.71. For cement-bone region 7 an agreement of 87.21-90.45% was found, which is a varied kappa(w) of 0.48-0.58. All the kappa values differ significantly from nil. The result shows that digital technique is as good as analog radiographs for diagnosing possible loosening of hip prostheses.

The physico-chemical characteristics of the phosphocholine-containing glycoglycerolipid MfGL-II govern the permeability properties of Mycoplasma fermentans.

Mycoplasma fermentans seems to be involved in several pathogenic conditions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycolipid 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-alpha-D-glucopyranosyl-(1'-->3)-1,2-diacylglycerol (MfGL-II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL-II were therefore investigated. When a cell culture ages the fraction of MfGL-II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL-II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico-chemical properties of MfGL-II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lalpha) phases is 35-45 degrees C higher for MfGL-II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL-II forms an Lalpha phase and isotropic aggregates which were interpreted to be vesicles with a radius of approximately 450 A. It is proposed that MfGL-II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL-II. Our results indicate that MfGL-II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.

Phase diagrams of systems with cationic alpha-helical membrane-spanning model peptides and dioleoylphosphatidylcholine.

Ternary phase diagrams have been constructed of systems with dioleoylphosphatidylcholine (DOPC) and water, and two alpha-helical membrane-spanning model peptides, KKLAKK16[KK(LA)6KK] and KKLAKK20[KK(LA)8KK]. It was found that these peptides induced non-lamellar liquid crystalline phases. The amount of peptide needed for this phase transition depended on the water content and the temperature; and for KKLAKK16, a smaller amount of peptide was needed to induce non-lamellar phases than for KKLAKK20. Both peptides were found to induce an isotropic phase, and KKLAKK16 also induced a reversed hexagonal phase. Both peptides may also reside in a lamellar (L(alpha)) phase. When magic angle spinning (MAS) 31P NMR experiments were performed on samples containing the L(alpha) phase and an isotropic phase, four different isotropic chemical shifts were observed. The isotropic chemical shifts could be assigned to the phases, using spinning sidebands to calculate the chemical shift anisotropy (CSA) corresponding to each isotropic shift. MAS 13C NMR also indicated a difference in the aggregational state of the peptides between the L(alpha) and isotropic phases. The phase diagrams were compared to the phase diagram of a similar model peptide, AWW(LA)5WWA in systems with DOPC and water. It was concluded that the phase behaviour was influenced by both electrostatic interactions between the peptides and the lipid headgroups, and the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer.

Influence of transmembrane peptides on bilayers of phosphatidylcholines with different acyl chain lengths studied by solid-state NMR.

The molecular orientation in a lipid membrane of the peptide fragment VEYAGIALFFVAAVLTLWSMLQYLSAAR (phosphatidylglycerophosphate synthase (Pgs) peptide E) of an integral membrane protein, Pgs, in Escherichia coli has been investigated by solid-state 15N nuclear magnetic resonance (NMR) on macroscopically aligned lipid bilayers. The secondary structure of the peptide in lipid vesicles was determined by circular dichroism spectroscopy. Furthermore, the phase behaviour of the Pgs peptide E/dierucoylphosphatidylcholine (DEruPC)/water system was determined by (2)H, (31)P and 15N solid-state NMR spectroscopy. The phase behaviour obtained was then compared to that of the Pgs peptide E solubilised in dioleoylphosphatidylcholine and water that was previously studied by Morein et al. [Biophys. J. 73 (1997) 3078-3088]. This was aimed to answer the question whether a difference in the length of the hydrophobic part of this peptide and the hydrophobic thickness of the lipid bilayer (hydrophobic mismatch) will affect the phase behaviour. The peptide mostly has a transmembrane orientation and is in an alpha-helical conformation. An isotropic phase is formed in DEruPC with high peptide content (peptide/lipid molar ratio (p/l) > or =1:15) and high water content (> or =50%, w/w) at 35 degrees C. At 55 and 65 degrees C an isotropic phase is induced at high water content (> or =50%, w/w) at all peptide contents studied (no isotropic phase forms in the lipid/water system under the conditions in this study). At high peptide contents (p/l> or =1:15) an isotropic phase forms at 20 and 40% (w/w) of water at 55 and 65 degrees C. A comparison of the phase behaviour of the two homologous lipid systems reveals striking similarities, although the thicknesses of the two lipid bilayers differ by 7 A. This suggests that the rationalisation of the phase behaviour in terms of the hydrophobic mismatch is not applicable to these systems. The C-terminus of Pgs peptide E is amphiphilic and a considerable part of the peptide is situated outside the hydrophobic part of the bilayer, a property of the peptide that to a large extent will affect the lipid/peptide phase behaviour.

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers.

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes.

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies.

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.

Total lipids with short and long acyl chains from Acholeplasma form nonlamellar phases.

The cell-wall-less bacterium Acholeplasma laidlawii A-EF22 synthesizes eight glycerolipids. Some of them form lamellar phases, whereas others are able to form normal or reversed nonlamellar phases. In this study we examined the phase properties of total lipid extracts with limiting average acyl chain lengths of 15 and 19 carbon atoms. The temperature at which these extracts formed reversed hexagonal (HII) phases differed by 5-10 degreesC when the water contents were 20-30 wt%. Thus the cells adjust the ratio between lamellar-forming and nonlamellar-forming lipids to the acyl chain lengths. Because short acyl chains generally increase the potential of lipids to form bilayers, it was judged interesting to determine which of the A. laidlawii A lipids are able to form reversed nonlamellar phases with short acyl chains. The two candidates with this ability are monoacyldiglucosyldiacylglycerol (MADGlcDAG) and monoglucosyldiacylglycerol. The average acyl chain lengths were 14.7 and 15.1 carbon atoms, and the degrees of acyl chain unsaturation were 32 and 46 mol%, respectively. The only liquid crystalline phase formed by MADGlcDAG is an HII phase. Monoglucosyldiacylglycerol forms reversed cubic (Ia3d) and HII phases at high temperatures. Thus, even when the organism is grown with short fatty acids, it synthesizes two lipids that have the capacity to maintain the nonlamellar tendency of the lipid bilayer. MADGlcDAG in particular contributes very powerfully to this tendency.

Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.

Occurrence and susceptibility to antibiotics of Shigella species in stools of hospitalized children with bloody diarrhea in Pakistan.

The aim of the present investigation was to study the frequency of Shigella spp. in patients with bloody diarrhea in Pakistan and the susceptibility of isolated Shigella to three antibiotics: ampicillin, cotrimoxazole and nalidixic acid. In addition, the frequency of Campylobacter and Salmonella was also determined. Stool samples (n = 152) were collected from 152 diarrheic children less than six years of age passing blood and mucus in their stools who were admitted to Paediatric Department of Mayo Hospital in Lahore, Pakistan from June to September 1990. The samples were cultivated on standard media for Shigella, Campylobacter, and Salmonella. Susceptibility of Shigella isolates was tested by disk diffusion method. The frequency of isolation was 19.1% for Shigella spp., 7.9% for Campylobacter, and 4.6% for Salmonella. Shigella flexneri (7.9%) was the most frequently isolated species, followed by S. dysenteriae (6.6%), S. boydii, (3.3%) and S. sonnei (1.3%). All Shigella isolates were susceptible to nalidixic acid (100%), while only a few were susceptible to cotrimoxazole (7.0%) and ampicillin (3.5%). In Pakistan, self-medication and purchases of drugs without a prescription are commonly practiced. Thus, there is a greater possibility of development of resistant strains due to over use of antibiotics.

Lipids in total extracts from Acholeplasma laidlawii A pack more closely than the individual lipids. Monolayers studied at the air-water interface.

Pressure-area curves were obtained at 25, 35 and 45 degrees C for total lipid extracts and four individual glucolipids isolated from Acholeplasma laidlawii strain A-EF22. The glucolipids are 1,2-diacyl-3-0-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDAG), 1,2 -diacyl-3-0-[alpha-D-glucopyranosyl-(1-->2)-0-alpha-D-glucopyranosyl] -sn-glycerol (DGlcDAG), 1,2-diacyl-3-0-[alpha-D-glucopyranosyl-(1-->2)-0-(6-0-acyl-alpha-D-gluco pyranosyl)]-sn-glycerol (MADGlcDAG), and 1,2-diacyl-3-0-[glycerophosphoryl-6-0-(alpha-D-glucopyranosyl-(1-- >)-0-alpha-D-glucopyranosyl)]-sn-glycerol (GPDGlcDAG). The total lipid extracts were obtained from A. laidlawii, grown at 37 degrees C with fatty acids of varying degrees of unsaturation and chain length. The mean surface area per molecule was obtained from these pressure-area curves at surface pressures equal to 10, 20, 30 and 40 mN/m. It was found that the interfacial area of the lipids increases with increasing degree of unsaturation, but is nearly independent of the acyl chain length at constant unsaturation. The surface charge density varied between 4.7 x 10(-3) e-/angstrom(2) and 9.4 x 10(-3) e-/angstrum(2) for the total lipid extracts studied, but did not exhibit any consistent dependence on variations in degree of unsaturation or acyl chain length. The mean area per molecule was found to be smaller for the total lipid extracts than for the individual lipids. It is concluded that the bacterium strives to regulate its lipid composition in such a way that the packing of the lipids in the membrane is appropriately tight, and/or to keep a slight negative spontaneous curvature of the lipid bilayer of the cell membrane ("optimal packing"). This is in accordance with the physico-chemical model for the regulation of the lipid composition in the membrane of A. laidlaiwii previously presented by us (see e.g. Andersson, A.-S., Riffors, L., Bergqvist, M., Persson, S. and Lindblom, G. (1996) Biochemistry 35, 11119-11130).

Occurrence of monoacyl-diglucosyl-diacyl-glycerol and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol in membranes of Acholeplasma laidlawii strain B-PG9.

It is shown by thin-layer and high-performance liquid chromatography that the two membrane lipids monoacyl-diglucosyl-diacyl-glycerol (MADGlcDAG) and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol are synthesized by Acholeplasma laidlawii strain B-PG9 when the cells are grown in two different growth media. The two lipids are also synthesized by A. laidlawii strain A-EF22 and their chemical structures have been determined previously by NMR spectroscopy. Since a reversed hexagonal phase is the only liquid-crystalline phase formed by MADGlcDAG, it is concluded that A. laidlawii strain B-PG9, in resemblance to strain A-EF22, synthesizes three membrane lipids that are able to form reversed nonlamellar phases. A comparison of the membrane lipids from the two strains shows that there is essentially one lipid from each strain that differs. However, both these lipids have common physico-chemical properties, namely the ability to form reversed nonlamellar phases. Finally, it is also shown that novel lipids may be synthesized by A. laidlawii through long-time adaptation to altered growth conditions.

Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system.

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.

Norfloxacin resistance in Campylobacter jejuni and Campylobacter coli isolates from Swedish patients.

This study focused on the frequency of Campylobacter jejuni and Campylobacter coli strains resistant to norfloxacin. Included were 1659 consecutive stool specimens isolated between 1992 and 1995, from as many Swedish patients with diarrhoea. The patients were divided with regard to place of infection and age. All strains were tested for susceptibility to norfloxacin by means of disc diffusion test on blood-agar plates. Norfloxacin-resistant strains (n = 310) were furthermore tested for resistance to doxycycline and erythromycin. The Etest was used for determination of MIC values of doxycyclin, erythromycin and norfloxacin of 81 of the strains. C. jejuni and C. coli isolations resistant to norfloxacin were significantly more frequent among patients infected abroad, especially in Spain and Thailand, compared with those infected in Sweden, adults more often than children. The number of resistant strains showed a yearly increase, and the difference between children and adults was equalized in 1995. The MIC50 and MIC90 values for doxycycline and erythromycin have increased markedly through the 4 years studied. This study shows that norfloxacin, because of increased resistance, may have limited utility for treatment of gastrointestinal infections caused by C. jejuni and C. coli.

Cryo-TEM and NMR studies of a micelle-forming phosphoglucolipid from membranes of Acholeplasma laidlawii A and B.

The chemical structure of a phosphoglucolipid from the membrane of the bacterium Acholeplasma laidlawii strain B-PG9 has been determined by high resolution NMR to be 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D-glucopyranosyl-(1 -->2)-O-alpha-D-glucopyranosyl)]-sn-glycerol (GPDGlcDAG). It was concluded that this lipid has exactly the same structure as one of the phosphoglucolipids from A. laidlawii strain A-EF22. By cryo transmission electron microscopy (cryo-TEM) and NMR diffusion techniques it was shown that, in highly diluted aqueous solutions, this membrane lipid forms long thread-like micelles in equilibrium with lipid vesicles. The cause of the occurrence of these different aggregates is discussed in terms of the varying molecular shapes of the lipid because of a heterogeneous composition of the acyl chains. A second membrane phosphoglucolipid from the bacterium, namely 1,2-diacyl-3-O-[glycerophosphoryl-6-O-(alpha-D- glucopyranosyl-(1 -->2)-monoacylglycerophosphoryl-6-O-alpha-D-glucopyranosyl)]-sn-gl ycerol (MABGPDGlcDAG), was found to form only a lamellar liquid crystalline phase coexisting with water.

Two-dimensional 1H-NMR of transmembrane peptides from Escherichia coli phosphatidylglycerophosphate synthase in micelles.

Two 28-residue peptides, PTLLTLFRVILIPFFVLVFYKKKGKKKG [Pgs-(6-25)-peptidyl-KKKGKKKG; Pgs peptide A] and VEYAGIALFFVAAVLTLWSMLQYLSAAR [Pgs-(149-176)-peptide, Pgs peptide E], were synthesized and studied by CD and two-dimensional 1H-NMR spectroscopy. The first 20 amino acid residues of Pgs peptide A are identical to one predicted transmembrane segment (Pro6-Tyr25) of the integral membrane protein phosphatidylglycerophosphate synthase (Pgs) of Escherichia coli. Pgs peptide E is identical to another predicted transmembrane segment (Val149-Arg176), which is located in the C-terminal end of this lipid synthase. Pgs peptides A and E were dissolved in methanol or trifluoroethanol or were incorporated into solvent-free micelles of fully deuterated SDS. In all these systems, CD spectra of both peptides indicated an alpha-helical secondary structure. However, peptides that were solubilized in micelles exhibited the highest content of alpha-helix as judged from comparison of the CD spectra. Thermodynamically stable isotropic solutions at high peptide concentrations (1-3 mM) could only be obtained with the peptide incorporated in micelles; in organic solvents, significant peptide aggregation occurred. Relatively sharp peaks were obtained with 1H-NMR spectroscopy of the peptides in SDS micelles, which indicates rapid tumbling of the peptides in the micellar environment. Translational-diffusion coefficients of the micelles with and without peptide, determined by pulsed-field-gradient NMR, showed that the micellar size was unaffected by the solubilized peptide. The radius of the hydrated micelles was estimated to be about 2.7 nm (i.e. the mass of the aggregate is almost 30 kDa). Two-dimensional NMR spectroscopy of both peptides solubilized in the micelles indicated an alpha-helical conformation. This observation is strengthened by an investigation of the hydrogen exchange of the peptide amide protons, where significantly less exchange of the amide protons was observed in the middle of the peptides compared with the ends.

New aspects on membrane lipid regulation in Acholeplasma laidlawii A and phase equilibria of monoacyldiglucosyldiacylglycerol.

A new membrane lipid, monoacyldiglucosyldiacylglycerol (MADGlcDAG), was recently discovered in Acholeplasma laidlawii strain A-EF22, demanding a new study of the biosynthetic regulation, and the phase behavior, of the glucolipids in this organism. The only liquid-crystalline phase formed by MADGlcDAG is a reversed hexagonal phase. A. laidlawii A-EF22 synthesizes four lipids that have the ability to induce the formation of reversed nonlamellar phases: MADGlcDAG, monoglucosyldiacylglycerol (MGlcDAG), monoacylmonoglucosyldiacylglycerol (MAMGlcDAG), and diacylglycerol (DAG). A Cn value of approximately 16 seems to be a critical value for the fractions of these lipids in the membrane: the fractions of MADGlcDAG and MGlcDAG are largest when the Cn values are lower than 16, while the fractions of MAMGlcDAG and DAG are largest when the Cn values are higher than 16. The fraction of nonlamellar-forming lipids was 55 mol% when the Cn value was 14.8 and the degree of unsaturation was 33 mol%. This fraction was reduced to 7 mol% when the Cn value and the degree of unsaturation were increased to 17.8 and 92 mol%, respectively, i.e., at conditions that markedly favor the formation of reversed nonlamellar phases. These observations convincingly show that a balance between lamellar- and nonlamellar-forming lipids is maintained in the membrane and strongly support the validity of the lipid regulation model proposed by us. From earlier biochemical data, obtained with short acyl chains, that were difficult to reconcile with our regulation model, it could be predicted that a lipid ought to be synthesized that assists MGlcDAG to maintain the nonlamellar-forming properties with the short chains. It is shown in the present work that this lipid is MADGlcDAG and that the regulation of the balance between lamellar- and nonlamellar-forming lipids is even more complex and sophisticated in A. laidlawii A-EF22 than previously proposed.

Wild-type Escherichia coli cells regulate the membrane lipid composition in a "window" between gel and non-lamellar structures.

Escherichia coli strain K12 was grown at 17, 27, and 37 degrees C. The acyl chain composition of the membrane lipids varied with the growth temperature; the fraction of cis-vaccenoyl chains decreased, and the fraction of palmitoyl chains increased, when the growth temperature was increased. However, the polar head group composition did not change significantly. The equilibria between lamellar and reversed non-lamellar phases of lipids extracted from the inner membrane (IM), and from both the membranes (IOM), were studied with NMR and x-ray diffraction. At temperatures above the growth temperature the lipid extracts formed a reversed hexagonal phase, or a bicontinuous cubic phase, depending on the degree of hydration of the lipids. It was observed that: 1) at equal elevations above the growth temperature, IM lipid extracts, as well as IOM lipid extracts, have a nearly equal ability to form non-lamellar phases; 2) IM extracts have a stronger tendency than IOM extracts to form non-lamellar phases; 3) non-lamellar phases are formed under conditions that are relatively close to the physiological ones; the membrane lipid monolayers are thus "frustrated"; and 4) as a consequence of the change of the acyl chain structures, the temperature for the lamellar gel to liquid crystalline phase transition is changed simultaneously, and in the same direction, as the temperature for the lamellar to non-lamellar phase transition. With a too large fraction of saturated acyl chains the membrane lipids enter a gel state, and with a too large fraction of unsaturated acyl chains the lipids transform to non-lamellar phases. It is thus concluded that the regulation of the acyl chain composition in wild-type cells of E. coli is necessary for the organism to be able to grow in a "window" between a lamellar gel phase and reversed non-lamellar phases.

Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans.

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.

Influence of monoglucosyldiacylglycerol and monoacylmonoglucosyldiacylglycerol on the lipid bilayer of the membrane from Acholeplasma laidlawii strain A-EF22.

The ability for 1,2-diacyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDAG) and 1,2-diacyl-3-O-(6-O-acyl-(alpha-D-glucopyranosyl))-sn-glycerol (MAMGlcDAG) to induce non-lamellar phases in a lipid mixture with an in vivo composition, prepared from Acholeplasma laidlawii membranes, has been investigated. The phase transition temperatures from lamellar to non-lamellar structures were studied with varying fractions of MGlcDAG and MAMGlcDAG. The transition temperature decreased from 73 +/- 2 degrees C for 20 mol% MGlcDAG to 43 +/- 1 degree C for 63 mol% MGlcDAG, in lipid mixtures where the other lipids are the native bilayer-forming lipids. MAMGlcDAG behaved differently and the phase transition temperatures were found to be almost constant and between 51-53 degrees C as the fraction of MAMGlcDAG varied between 11-45 mol%. It was also found that MAMGlcDAG can only be solubilized in low concentrations in the lipid bilayer, which is in good agreement with the fractions of MAMGlcDAG found in the membrane of A. laidlawii. Higher concentrations of MAMGlcDAG resulted in phase separations of lamellar liquid crystalline and gel/crystalline phases. It is concluded that MAMGlcDAG is far more capable than MGlcDAG to induce non-lamellar structures at lower concentrations. The results are discussed in terms of the model of lipid regulation previously proposed by this laboratory (Lindblom, G., Hauksson, J.B., Rilfors, L., Bergenståhl, B., Wieslander, A. and Eriksson, P.O. (1993) J. Biol. Chem. 268, 16198-16207), and the importance for the bilayer stability in cell membranes. It is proposed that the phase behaviour of the membrane lipids has far-reaching consequences for membrane function.