Combined force spectroscopy, AFM and calorimetric studies to reveal the nanostructural organization of biomimetic membranes.

In this work we studied a binary lipid matrix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), a composition that mimics the inner membrane of Escherichia coli. More specifically, liposomes with varying fractions of POPG were analysed by differential scanning calorimetry (DSC) and a binary phase diagram of the system was created. Additionally, we performed atomic force microscopy (AFM) imaging of supported lipid bilayers (SLBs) of similar compositions at different temperatures, in order to create a pseudo-binary phase diagram specific to this membrane model. AFM study of SLBs is of particular interest, as it is conceived as the most adequate technique not only for studying lipid bilayer systems but also for imaging and even nanomanipulating inserted membrane proteins. The construction of the above-mentioned phase diagram enabled us to grasp better the thermodynamics of the thermal lipid transition from a gel-like POPE:POPG phase system to a more fluid phase system. Finally, AFM force spectroscopy (FS) was used to determine the nanomechanics of these two lipid phases at 27°C and at different POPG fractions. The resulting data correlated with the specific composition of each phase was calculated from the AFM phase diagram obtained. All the experiments were done in the presence of 10 mM of Ca(2+), as this ion is commonly used when performing AFM with negatively charged phospholipids.

Improving ex vivo skin permeation of non-steroidal anti-inflammatory drugs: enhancing extemporaneous transformation of liposomes into planar lipid bilayers.

Transdermal delivery of active principles is a versatile method widely used in medicine. The main drawback for the transdermal route, however, is the low efficiency achieved in the absorption of many drugs, mostly due to the complexity of the skin barrier. To improve drug delivery through the skin, we prepared and characterized liposomes loaded with ibuprofen and designed pharmaceutical formulations based on the extemporaneous addition of penetration enhancer (PE) surfactants. Afterwards, permeation and release studies were carried out. According to the permeation studies, the ibuprofen liposomal formulation supplemented with PEs exhibited similar therapeutic effects, but at lower doses (20%) comparing with a commercial formulation used as a reference. Atomic force microscopy (AFM) was used to investigate the effect caused by PEs on the adsorption mechanism of liposomal formulations onto the skin. Non-fused liposomes, bilayers and multilayered lipid structures were observed. The transformation of vesicles into planar structures is proposed as a possible rationale for explaining the lower doses required when a liposome formulation is supplemented with surfactant PEs.

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

Determination of the partition coefficients of a homologous series of ciprofloxacin: influence of the N-4 piperazinyl alkylation on the antimicrobial activity.

Partitioning of a fluoroquinolone antibiotic, ciprofloxacin, and its N-piperazinyl alkyl derivatives, between octanol or Escherichia coli lipid membrane extract and aqueous buffer pH 7.4, was studied. The experimental partition coefficients (Pexp) were corrected at this pH using an expression that includes the microconstant values of each compound. The relationship between the corrected partition coefficients expressed as logP (thermodynamic partition coefficient) and the diffusion through the lipid bilayers ('hydrophobic pathway') of entry has been considered here. In this work, we have explored the possibility of using our model to provide physicochemical evidences to support such a via. The correlation between logP values and antibacterial activities (expressed as minimal inhibitory concentration (MIC) values) of the homologous series of antibiotics against different bacteria were studied. A parabolic behaviour was observed which evidenced that the only increase in lipophilicity does not result in an enhanced antimicrobial activity for the homologous family studied.

Determination by fluorimetric titration of the ionization constants of ciprofloxacin in solution and in the presence of liposomes.

A fluorescence titration method was applied for the determination of pKa of ciprofloxacin (CPX) in solution. Values of 6.18 +/- 0.05 and 8.76 +/- 0.03 were obtained for pKa1 and pKa2, respectively. The method was used to determine the ionization constants in the presence of liposomes of dipalmitoylphosphatidylcholine (DPPC) and DPPC with 10 mol% of dipalmitoylphosphatidylglycerol. A dependence on the surface charge of liposomes was found which supported the existence of a basic electrostatic interaction between CPX and the phospholipid bilayer. Both pK values for the N-4 butyl-piperazinyl derivative (BCPX) of the parent compound were also determined in solution and in the presence of liposomes. The competition of both drugs for the same binding site as 1-anilino-8-naphtalene sulfonate demonstrate that the interaction is governed by electrostatic forces.

Evidence of an efflux pump in Serratia marcescens.

Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10(-7) to 10(-9). Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug.

Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells.

T cells are prominent components of both early and late atherosclerotic lesions and the role of Th1/Th2 cells subsets in the evolution and rupture of the plaque is currently under investigation. Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exert actions beyond that of simply lowering cholesterol levels, and some effects on immune function have been reported. We studied in vitro the effects of fluvastatin on Th1/Th2 cytokine release in relation to caspase-1 activation, in human peripheral-blood mononuclear cells (PBMC) stimulated or not with Mycobacterium tuberculosis. Fluvastatin treatment resulted in the activation of caspase-1 and in a small secretion of interleukin (IL)-1beta, IL-18, and IFNgamma (Th1). In the presence of bacteria, the release of these cytokines was highly increased by the statin in a synergistic way. By contrast, production of IL-12, IL-10 and IL-4 were unaffected by the statin. Not only did mevalonate abolish the effects of the statin but it also prevented the caspase-1 activation induced by the bacteria, suggesting the involvement of isoprenoids in the response to M. tuberculosis. It is proposed that inhibition of HMG-CoA reductase may be immunoprotective by enhancing the Th1 response, which has therapeutical potential not only in atherosclerosis but also in infectious diseases.

Characterization of allergens from Trisetum paniceum pollen: an important aeroallergen in Mediterranean continental climatic areas.

BACKGROUND: Trisetum paniceum is a grass plant which is characteristic of a Mediterranean continental climate and has been described as one of the major causes of type I allergy in the Madrid region. OBJECTIVES: To identify and characterize the allergens of Trisetum paniceum pollen. METHODS: Allergenic extracts were prepared by 24 h incubation of pollens in a buffered solution. Proteins were analysed by a new two-dimensional system in which agarose plates were used for isoelectric focusing. Two-dimensionally resolved proteins were electrically transferred to Immobilon membranes and the allergens immunochemically detected. Proteins from six grass pollens were bound to a membrane and incubated with a pool of serum from grass-pollen-sensitized patients. The bound IgE antibodies were then eluted and used to identify the proteins of Trisetum paniceum pollen that allergenically crossreact with allergens from other pollen grasses. RESULTS: Relative to total protein content, Trisetum paniceum pollen had a high proportion of reactive proteins. On the basis of their molecular characteristics, allergens could be classified as group 1, 2, 4 and 5 components yet included an atypical proportion of basic components. All identified allergens were crossreactive with allergens from the remaining grass pollens studied. CONCLUSIONS: Trisetum paniceum pollen contains a high proportion of allergens and these include a group of basic proteins which are not detected in other phylogenetically related pollens and could be of allergological interest.

Encapsulation of a quinolone in liposomes. Location and effect on lipid bilayers.

The effect of a new fluoroquinolone on the distearoyl phosphatidylcholine (DSPC) bilayers was examined above and below the phase transition temperature (Tm) of the lipid. It was found by photon correlation spectroscopy that size and polydispersity of the extruded liposomes were unaffected by quinolone. Moreover, fluorescence quenching methods revealed a low fraction (13%) of non-accessible population of drug in the vesicles. This was interpreted in terms of encapsulation efficiency. However, variations in size correlated with decrease in the values of precipitation factor (P). These results reveal the instability of quinoline-DSPC liposomes beyond 5 days.

Allergens from rye pollen (Secale cereale). I. Study of protein release by rye pollen during a 19-hour extraction process. Allergen identification.

We have studied the proteins and allergens released by rye pollen in the course of a 19-h pollen incubation process. Nearly 40% of the total extracted proteins were collected during the first 5 min, and most of them had a molecular weight less than 28 kDa. Between 5 and 30 min, 15% of the proteins from total extract were released, showing in the SDS-PAGE analysis an increase in which components moved close to 30 kDa standard. From 30 min to 19 h several extracts were collected. Electrophoretical profile of components from these extracts reveals that bands moving below 28 kDa were practically absent and those of 28 and 23 kDa became very intense. At the end of the process there was a rise of 67 kDa proteins. Dot-immunobinding and immunoblotting techniques reveal that allergens leave the rye pollen, for the most part, after 5 min incubation and are proteins with 28 kDa, 33 kDa, 48 kDa and 67 kDa molecular weights.

Allergens from rye pollen (Secale cereale). II. Characterization and partial purification.

Rye pollen was incubated for 30 min and proteins extracted at this time were collected as extract A (EA). The same pollen grains were resuspended in buffer and incubated for 18.5 h. Proteins extracted in this period were designated extract B (EB). Both extracts were subfractionated by DEAE ion-exchange chromatography and allergen presence in peaks detected by the dot-immunobinding technique. The results reveal that unretained proteins (peaks 1 and 2) and proteins eluted at 0.2 M NaCl from extract B contain the highest proportion of allergens. SDS-PAGE of chromatographic peaks showed that peak 2 from extract B contains a highly purified 28 kDa band. On the skin of allergic patients this band gave a stronger positive prick test than for the crude extract.

Serosurveillance of susceptibility to rubella in a Mexican female group.

The susceptibility to rubella of a group of Mexican females of childbearing age was determined. Members of the group were selected based on the highest probability of having been exposed to the virus they were older than 15, and from a low socio-economic urban stratum. Anti-viral antibodies were determined by hemagglutination inhibition. Concentration was expressed as International Units of IgG anti-rubella hemagglutinin (IU). Antibody concentrations lower than 15.6 IU/ml were regarded as non-protective. A 16% susceptibility was found.

A method for culturing rat oviduct gamma-aminobutyric acid cells.

A technique for the culture of rat oviduct gamma-aminobutyric acid (GABA) cells is described. The technique involves first explaining the fimbria and preampulla, which are the oviduct divisions with the highest density of GABA cells. The explanted tissue is cultured in a serum-free medium, to propagate the outgrowing cells. Under the experimental conditions we describe, the majority of the cells maintain GABA expression, as determined by immunostaining with a GABA antiserum.

Serologically proven acute rubella infection in patients with clinical diagnosis of dengue.

Patients with a clinical diagnosis of dengue but negative by serological testing were studied for rubella infection. Paired sera were obtained from 69 patients during an outbreak in Yucatán, México. The presence of specific anti-viral IgM in the acute sera was considered as diagnostic for rubella infection. The immunoglobulin was determined by measuring the difference in the inhibition of hemagglutination between the non-reduced and the reduced fractionated sera. Immunoglobulins were separated by sucrose density centrifugation. Acute rubella infection was found in 7 (10.1%) of the patients. These results demonstrate active rubella infection in patients clinically diagnosed as dengue.

Physicochemical properties of a human glycoprotein bearing blood group A activity.

A human erythrocyte glycoprotein was isolated and purified from blood of group A+1 by a procedure involving chloroform-methanol extraction and affinity chromatography on Helix pomatia lectin-Sepharose 6MB, and some of its physicochemical properties were determined. The resulting preparation was homogeneous as indicated by polyacrylamide gel electrophoresis. The glycoprotein contained nearly 60% carbohydrate and 40% protein. It was water-soluble and inhibited the agglutination of A-erythrocytes. Its molecular weight was 41,900 (amino acid analysis) or 55,200 (light scattering), whereas electrophoresis revealed two bands of 43,000 and 76,000 Da. The ORD spectrum was consistent with 30% alpha-helix, 20% beta-sheet, and 50% random coil. Intrinsic viscosity was 14.61 ml.g-1, partial specific volume was roughly 0.66, isoelectric and isoionic points were 6.90 and 6.95, respectively. The glycoprotein differs from glycophorin and appears to be one of the minor glycoproteins of the human erythrocyte membrane.

A re-examination of the Na+-independent binding of [3H]beta-alanine to rat brain stem-spinal cord.

The Na+-independent binding of [3H]beta-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described beta-alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na+-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na+-independent binding, using frozen/thawed/washed synaptosomal-mitochondrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200-415 micrograms/ml incubation medium. Binding was completely inhibited by glycine, alanine, alpha-aminobutyric acid, beta-aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl-beta-alanine, brucine and gelsemine. It was insensitive to taurine, gamma-aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (KD 20 microM), and heat sensitive.

Detection of cell surface rubella virus antigens in Vero cells with Staphylococcus aureus rich in protein A.

The presence of cell surface rubella antigen was used to verify and monitor viral replication in Vero cell monolayers. Viral antigen was observed in infected cells by the adherence of Staphylococcus aureus sensitized with immune anti-rubella sera. The staphylococci specifically bound to infected cells were Gram-stained and observed using light microscopy. The minimum titer of IgG antiviral hemagglutinin required for sensitizing the bacteria was 3.9 IU/ml. The specificity of the assay was demonstrated by treating the infected cells with bacteria sensitized with normal sera, by treating the mock-infected cells with staphylococci sensitized with either immune or normal sera, and by sensitizing the bacteria with immune sera from which anti-rubella antibodies had been removed. Viral antigens were detected from day 2-9 post-infection. The sensitivity of the assay in verifying and monitoring viral propagation was comparable to the titration of viral particles of hemagglutination. The assay is specific, rapid, simple and can be performed in laboratories with minimal equipment.

On the binding of cinmetacin and indomethacin to human serum albumin.

The binding of two non-steroidal anti-inflammatory drugs, indomethacin and cinmetacin, to human serum albumin was studied by dynamic dialysis at 37 degrees C and pH 7.4. Cinmetacin is bound more than indomethacin. The affinity constant for the primary binding site is 4.28 X 10(6) M-1 for cinmetacin and 1.4 X 10(6) M-1 for indomethacin. The protein binding of indomethacin is decreased in the presence of cinmetacin.

[3H]prazosin binding to central nervous system regions of male and female rats.

Membranes were obtained from the pituitary gland and 13 cerebral regions of male rats and of females during two endocrinological states (estrous and diestrous-1), and assayed for alpha 1-receptor binding, using [3H]prazosin. At two [3H]prazosin concentrations, one near its binding dissociation constant and the other at a value at which the plateau was reached in the binding saturation curve (0.2 and 1.2 nM respectively), [3H]prazosin binding was highest to the frontal cortex, lowest to the spinal cord, and showed no differences in any region under any of the hormonal states tested. Results are discussed in relation to previously reported hormonal effects on alpha 1-receptors.