Maintenance of bacterial outer membrane lipid asymmetry: insight into MlaA.

The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified - and called VacJ - based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections.

The molecular mechanism of Nystatin action is dependent on the membrane biophysical properties and lipid composition.

Nystatin (Nys) is a pore forming broad-spectrum and efficient antifungal drug with significant toxicity in mammalian organisms. In order to develop a non-toxic and more effective Nys formulation, its molecular mechanism of action at the cell membrane needs to be better understood. It is widely accepted that Nys activity and toxicity depend on the presence and type of membrane sterols. Taking advantage of multiple biophysical methodologies, we now show that the formation and stabilization of Nys aqueous pores, which are associated with Nys cytotoxicity, occur in the absence of membrane sterols. Our results suggest that the Nys mechanism of action is driven by the presence of highly ordered membrane domains capable of stabilizing the Nys oligomers. Moreover, Nys pore formation is accompanied by strong Nys-induced membrane reorganization that depends on membrane lipid composition and seems to underlie the Nys cytotoxic effect. Accordingly, in membranes enriched in a gel-phase forming phospholipid, Nys incorporates within the phospholipid-enriched gel domains, where it forms pores able to expand the gel domains. In contrast, in membranes enriched in gel domain forming sphingolipids, Nys-induced pore formation occurs through the destabilization of the gel phase. These results show that the Nys mechanism of action is complex and not only dependent on membrane sterols, and provide further insight into the molecular details governing Nys activity and toxicity.

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: a new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-ß-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-ß-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

Serological follow-up in patients with aorto-iliac disease and evidence of Q fever infection.

The aim of this study was to provide data on the risk of developing chronic Q fever in patients with aorto-iliac disease and evidence of previous Q fever infection. Patients with an aortic and/or iliac aneurysm or aorto-iliac reconstruction (aorto-iliac disease) and evidence of previous Q fever infection were included. The presence of phase I and II Coxiella burnetii IgG antibodies was assessed periodically using immunofluorescence assay. A total of 111 patients with aorto-iliac disease were divided into three groups, based upon the serological profile [mean follow-up: 16 ± 9 months (mean ± standard deviation)]. Group 1 consisted of 30 patients with a serological trace of C. burnetii infection (negative IgG phase I, IgG phase II titer of 1:32). Of these, 36.7% converted to serological profile matching past resolved Q fever. Group 2 included 49 patients with negative IgG phase I titer and IgG phase II titer ≥1:64. No patients developed chronic Q fever, but 14.3% converted to a positive IgG phase I titer. Group 3 consisted of 32 patients with positive IgG phase I and positive IgG phase II titers, of which 9.4% developed chronic Q fever (significantly different from group 2, p = 0.039). The IgG phase I titer increased in 28.1% of patients (from 1:64 to 1:4,096). The risk of developing chronic Q fever in patients with aorto-iliac disease and previous Q fever infection with a positive IgG phase I titer was 9.4%. The IgG phase I titer increases or becomes positive in a substantial number of patients. A standardized serological follow-up is proposed.

Insight into the outer membrane asymmetry of P. aeruginosa and the role of MlaA in modulating the lipidic composition, mechanical, biophysical, and functional membrane properties of the cell envelope.

In Gram-negative bacteria, the outer membrane (OM) is asymmetric, with lipopolysaccharides (LPS) in the outer leaflet and glycerophospholipids (GPLs) in the inner leaflet. The asymmetry is maintained by the Mla system (MlaA-MlaBCDEF), which contributes to lipid homeostasis by removing mislocalized GPLs from the outer leaflet of the OM. Here, we ascribed how Pseudomonas aeruginosa ATCC 27853 coordinately regulates pathways to provide defense against the threats posed by the deletion of mlaA. Especially, we explored (i) the effects on membrane lipid composition including LPS, GPLs, and lysophospholipids, (ii) the biophysical properties of the OM such as stiffness and fluidity, and (iii) the impact of these changes on permeability, antibiotic susceptibility, and membrane vesicles (MVs) generation. Deletion of mlaA induced an increase in total GPLs and a decrease in LPS level while also triggering alterations in lipid A structures (arabinosylation and palmitoylation), likely to be induced by a two-component system (PhoPQ-PmrAB). Altered lipid composition may serve a physiological purpose in regulating the mechanobiological and functional properties of P. aeruginosa. We demonstrated an increase in cell stiffness without alteration of turgor pressure and inner membrane (IM) fluidity in ∆mlaA. In addition, membrane vesiculation increased without any change in OM/IM permeability. An amphiphilic aminoglycoside derivative (3',6-dinonyl neamine) that targets P. aeruginosa membranes induced an opposite effect on ∆mlaA strain with a trend toward a return to the situation observed for the WT strain. Efforts dedicated to understanding the crosstalk between the OM lipid composition, and the mechanical behavior of bacterial envelope, is one needed step for designing new targets or new drugs to fight P. aeruginosa infections.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium responsible for severe hospital-acquired infections. The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore, compromising this structure could increase sensitivity to antibiotics. The OM is asymmetric with the highly packed lipopolysaccharide monolayer at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. In this study, we show that deleting mlaA, the membrane component of Mla system located at the OM, affects the mechanical and functional properties of P. aeruginosa cell envelope. Our results provide insights into the role of MlaA, involved in the Mla transport pathway in P. aeruginosa.

Deficient Pseudomonas aeruginosa in MlaA/VacJ outer membrane lipoprotein shows decrease in rhamnolipids secretion, motility, and biofilm formation, and increase in fluoroquinolones susceptibility and innate immune response.

Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital acquired infections poses threat by its ability for adaptation to various growth modes and environmental conditions and by its intrinsic resistance to antibiotics. The latter is mainly due to the outer membrane (OM) asymmetry which is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. It comprises six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids mislocalized at the outer leaflet of OM. To investigate the role of P. aeruginosa OM asymmetry especially MlaA, this study investigated the effect of mlaA deletion on (i) the susceptibility to antibiotics, (ii) the secretion of virulence factors, the motility, biofilm formation, and (iii) the inflammatory response. mlaA deletion in P. aeruginosa ATCC27853 results in phenotypic changes including, an increase in fluoroquinolones susceptibility and in PQS (Pseudomonas Quinolone Signal) and TNF-α release and a decrease in rhamnolipids secretion, motility and biofilm formation. Investigating how the mlaA knockout impacts on antibiotic susceptibility, bacterial virulence and innate immune response will help to elucidate the biological significance of the Mla system and contribute to the understanding of MlaA in P. aeruginosa OM asymmetry.

Behçet's disease uveitis.

Uveitis in Behçet's disease (BD) is frequent (40% of cases) and is a major cause of morbidity. The age of onset of uveitis is between 20 and 30 years. Ocular involvement includes anterior, posterior or panuveitis. It is non-granulomatous. Uveitis may be the first sign of the disease in 20% of cases or it may appear 2 or 3 years after the first symptoms. Panuveitis is the most common presentation and is more commonly found in men. Bilateralisation usually occurs on average 2 years after the first symptoms. The estimated risk of blindness at 5 years is 10-15%. BD uveitis has several ophthalmological features that distinguish it from other uveitis. The main goals in the management of patients are the rapid resolution of intraocular inflammation, prevention of recurrent attacks, achievement of complete remission, and preservation of vision. Biologic therapies have changed the management of intraocular inflammation. The aim of this review is to provide an update previous article by our team on pathogenesis, diagnostic approaches, identification of factors associated with relapse and the therapeutic strategy of BD uveitis.

[Identification of factors associated with performance in objective structured clinical examination in Rouen medical school]. / Identification des facteurs associés à la réussite aux examens cliniques objectifs et structurés dans la faculté de médecine de Rouen.

CONTEXT: Objective structured clinical examination (OSCE) became a national exam at the end of medical studies in France. The aim of this study was to identify the predictive factors for success at OSCEs. METHODS: Aurvey query after the OSCEs was completed by fifth-year medicine students at Rouen Uuniversity.. Data on continuous variables were compared using the Mann-Whitney test. Data on quantitative variables were compared using the Spearman's correlation. RESULTS: Two hundred and thirty-nine students, i.e., 98.7 % of the students, responded to the query. The median (IQR 25-75) OSCE score was 13.6/20 (12.5-14.2). Students' personal factors significantly associated with a higher OSCE performance were female sex (median score of 13.7 versus 13.4; P=0.03) and good health during the clerkship (median score of 13.6 versus 12.6; P=0.02). A higher OSCE performance was associated with an increased number (≥6)  of medicine clerkships (median score of 13.8 versus 13.3; P=0.02) and a decreased number (<3) of surgery clerkships (median score of 13.7 versus 12.9; P=0.009). There was no correlation between the OSCE score and medical school performance (Spearman's correlation, r=0.24). CONCLUSION: Homogenization of student's clerkships, assistance to students with health problems seem to be teaching approaches to promote success at OSCEs.

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin.

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Financial consequences of hospital-acquired bacteraemia in three Belgian hospitals in 2003 and 2004.

The financial and human costs of hospital-acquired infections are increasingly recognised in many healthcare systems. This study seeks to quantify excess expenditures on hospital-acquired bacteraemia (HAB) in three Belgian general hospitals in 2003 and 2004. Patients with HAB were compared with patients in the same All Patient Refined Diagnosis Related Groups (APR-DRGs) without HAB. Patient level costs were estimated using a hospital costing system developed by the 'Université Libre de Bruxelles', and compared with DRG-based funding for the three hospitals. HAB incidence was consistent with the national rate for two of the three hospitals, but considerably higher for the third. Both severity of illness and mortality were higher in the HAB group. Nosocomial bacteraemia was associated with an increased length of stay of 30 days and of 6.1 days in intensive care units. When compared with uninfected patients in the same DRG, treatment of HAB patients cost an additional euro 16,709. At current funding rates, hospitals made a mean profit of euro 446 for uninfected patients, but a mean loss of euro 2,431 for patients with HAB. Our findings suggest that hospitals have a financial interest in reducing the rate of HAB, even in a system which funds such complications through severity adjustments in the APR-DRG system. Growing international interest in pay for performance and other funding schemes will only strengthen these financial incentives.

[Additional non-contraceptive effects of contraception: CNGOF Contraception Guidelines]. / Bénéfices non contraceptifs des contraceptions. RPC Contraception CNGOF.

Hormonal and intrauterine contraceptive methods provide women with highly efficient protection against undesired pregnancy. Additional non-contraceptive effects are now well documented. Combined hormonal contraceptives use, either through the oral transdermal and vaginal route, allow a reduction in menorrhagia, dysmenorrhea, functional ovarian cysts, benign breast and uterine disease, endometriosis-related pain and recurrence. A reduction in ovarian cancer risks, including in women with BRCA syndrome, endometrial and colon cancer is documented. This effect is prolonged for years after contraception discontinuation. Non-contraceptive benefits of progestin-only contraceptives are less documented. Use of the levonorgestrel IUD is associated with a reduction in menorrhagia, dysmenorrhea including in case of endometriosis. Copper IUD use is associated with a decrease in cervix and endometrial cancer risk.

Fate of endocrine disrupters in waste stabilization pond systems.

The performance in the removal of estrogenicity from wastewater was studied in three wastewater treatment plants (WWTPs). Different treatment processes were evaluated: stabilization ponds and trickling filter. Sampling was performed from the input to the output of the treatment systems. The total estrogenic activity was determined with MCF-7-derived cell lines which express the endogenous estrogen receptor alpha. The two wastewater stabilization ponds with long retention time had high removal of estrogenicity (90% to 95%). Trickling filters despite being effective at removing organic load were less effective in removing estrogenicity (42%), and post tertiary ponds enhanced estrogenicity removal.

Contribution of plasma membrane lipid domains to red blood cell (re)shaping.

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers.

Azithromycin is a macrolide antibiotic known to bind to lipids and to affect endocytosis probably by interacting with lipid membranes [Tyteca, D., Schanck, A., Dufrene, Y.F., Deleu, M., Courtoy, P.J., Tulkens, P.M., Mingeot-Leclercq, M.P., 2003. The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages. J. Membr. Biol. 192, 203-215]. In this work, we investigate the effect of azithromycin on lipid model membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Thermal transitions of both lipids in contact with azithromycin are studied by (31)P NMR and DSC on multilamellar vesicles. Concerning the DPPC, azithromycin induces a suppression of the pretransition whereas a phase separation between the DOPC and the antibiotic is observed. For both lipids, the enthalpy associated with the phase transition is strongly decreased with azithromycin. Such effects may be due to an increase of the available space between hydrophobic chains after insertion of azithromycin in lipids. The findings provide a molecular insight of the phase merging of DPPC gel in DOPC fluid matrix induced by azithromycin [Berquand, A., Mingeot-Leclercq, M.P., Dufrene, Y.F., 2004. Real-time imaging of drug-membrane interactions by atomic force microscopy. Biochim. Biophys. Acta 1664, 198-205] and could help to a better understanding of azithromycin-cell interaction.

Humoral antibody response to bovine leukemia virus infection in cattle and sheep.

In this study, 345 cattle from 7 herds with a history of lymphosarcoma were tested for antibody to BLV antigens by three serological methods, namely immunodiffusion using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 as antigen, and radioimmunoassay using a bovine leukemia virus glycoprotein with a molecular weight of 60,000 and a bovine leukemia virus protein with a molecular weight of 24,000 as antigen. The three tests under comparison agreed for 335 animals, 240 being negative in the three tests, and 95 being positive. Results were variable in ten cases only. Glycoprotein with a molecular weight of 60,000 antibody titers were systematically higher than were protein with a molecular weight of 24,000 antibody titers in bovine sera and milk, as well as in sera of experimentally infected sheep. In the latter case, antibodies to bovine leukemia virus antigens reached maximal values at the animal death in the tumor phase of the disease. Ratios of serum antiglycoprotein titer to milk titer varied between 4 and 117, showing that, if milk pools are to be used in surveys of bovine leukemia virus infection, use of very sensitive techniques of detection is mandatory.

Value of 18F-FDG PET/CT in diagnosing chronic Q fever in patients with central vascular disease.

BACKGROUND: The aim of this study is to describe the value of 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) in diagnosing chronic Q fever in patients with central vascular disease and the added value of 18F-FDG PET/CT in the diagnostic combination strategy as described in the Dutch consensus guideline for diagnosing chronic Q fever. METHODS: 18F-FDG PET/CT was performed in patients with an abdominal aortic aneurysm or aorto-iliac reconstruction and chronic Q fever, diagnosed by serology and positive PCR for Coxiella burnetii DNA in blood and/or tissue (PCR-positive study group). Patients with an abdominal aortic aneurysm or aorto-iliac reconstruction without clinical and serological findings indicating Q fever infection served as a control group. Patients with a serological profile of chronic Q fever and a negative PCR in blood were included in additional analyses (PCR-negative study group). RESULTS: Thirteen patients were evaluated in the PCR-positive study group and 22 patients in the control group. 18F-FDG PET/CT indicated vascular infection in 6/13 patients in the PCR-positive study group and 2/22 patients in the control group. 18F-FDG PET/CT demonstrated a sensitivity of 46% (95% CI: 23-71%), specificity of 91% (95% CI: 71-99%), positive predictive value of 75% (95% CI:41-93%) and negative predictive value of 74% (95% CI: 55-87%). In the PCR-negative study group, 18F-FDG PET/CT was positive in 10/20 patients (50%). CONCLUSION: The combination of 18F-FDG PET/CT, as an imaging tool for identifying a focus of infection, and Q fever serology is a valid diagnostic strategy for diagnosing chronic Q fever in patients with central vascular disease.

The different electrophoretic forms of post gamma-globulin their antigenic identity and their structural variability.

Post gamma-globulin, first described as a constant component of protein of cerebrospinal fluid and urine from patients with tubular disorders has also been found in other biological fluids. Post gamma-globulin from a single individual always migrated as several bands after storage. Three electrophoretic forms, immunochemically identical, have been isolated by gel chromatography, preparative continuous flow electrophoresis and ion-exchange chromatography. Their molecular weight was found to be approximately between 11 000 to 12 000. No difference between the three forms could be detected. The N-terminal amino acids were found to be Lys, Arg and Leu respectively for the three forms of post gamma-globulin. The "slow" and "fast" forms of post gamma-globulin seemed to differ by elimination of small basic peptides or amino acids from the N-terminal end of the protein. No enzymatic activity of post gamma-globulin was found, but this requires further investigations.

Real-time imaging of drug-membrane interactions by atomic force microscopy.

Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.