Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.     

Apoptosis signalling is essential and precedes protein degradation in wasting skeletal muscle during catabolic conditions

Activation of skeletal muscle proteolysis leads to wasting in many types of catabolic/chronic diseases. Protein breakdown is basically accomplished by the activation of the ubiquitin-proteasome system. Interestingly, several publications have shown that DNA fragmentation also occurs in skeletal muscle tissue during catabolism. The present review suggests that activation of apoptosis precedes protein breakdown associated with muscle wasting. In addition, the role of the different proteolytic systems and their relation with apoptosis is emphasized. Altogether, the data presented could be used for the design of new approaches for the treatment of muscle wasting diseases.     

A kinetic method for quantification of aspartate aminotransferase isoenzymes

A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.     

Effects of a fungal lipase on membrane organization evaluated by fluorescence polarization

Triglyceride lipase from Thermomyces lanuginosa (TlL) binds to the non-substrate small (40 nm) unilamelar vesicles of 1,2-dimiristoylglycero-sn-3-phosphoglycerol (DMPG-SUVs) in a catalytically active structure, whereas it adopts a catalytically incompetent form in binding to zwitterionic 1,2-dimiristoylglycero-sn-3-phosphocholine (DMPC-SUVs) or to large (100 nm) unilamelar DMPG (DMPG-LUVs) vesicles. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of TlL (and its mutants) on the mobility of the phospholipids. All TlL mutants insert into the DMPG-SUVs and increase lipid order at the headgroup region and at the hydrophobic core of the lipid bilayer as well. The increase of the rigidity of the membrane that occurs in the gel and liquid crystal states, results in an increase of the phase transition temperature (T_m). Kinetic experiments with monolayers of 1,2-dicaprin demonstrate the thermal stability of the enzyme in the range of temperatures of the phase transition. Mutations in the tryptophan (Trp) residues of TlL reduce activity of this enzyme and affect its interaction with the membrane. The membrane insertions of TlL mutants with other than Trp substitutions are much more shallower and produce only small increases of T_m, whereas mutation of lid-located Trp89 or mutation of any other Trp residue (117, 221, 260) result in a deeper penetration and significant increases of the T_m. Lipid dynamics of DMPC-SUVs or DMPG-LUVs are not affected by any of the TlL mutants, despite their strong binding to the lipids revealed by resonance energy transfer (RET). These results are consistent with the lipase–lipid penetration model in which the “lid” region of TlL inserts into the highly curved anionic interface, thus stabilizing the “open” or active enzyme conformation, whereas TlL binds to the surface of zwitterionic and large (small curvature) anionic vesicles in a “closed” (or inactive) conformation, without insertion of the lid.     

Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils

World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.     

Genotypic and Phenotypic Applications for the Differentiation and Species-Level Identification of Achromobacter for Clinical Diagnoses

The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected ‘house-keeping’ genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.     

In the rat, tumor necrosis factor α administration results in an increase in both UCP2 and UCP3 mRNAs in skeletal muscle: a possible mechanism for cytokine-induced thermogenesis?

Since the discovery of the new members of the UCP (uncoupling protein) family, UCP2 and UCP3, very few studies have dealt with the regulation of their expression. Bearing this in mind, administration of a single intravenous injection of TNF-α (100 μg/kg body weight) to rats resulted in a significant increase in UCP2 (242%) and UCP3 (113%) gene expression in skeletal muscle. The results suggest a possible role for UCP2 and UCP3 in the increase of energy expenditure associated with cytokine treatment.     

Activation of UCPs gene expression in skeletal muscle can be independent on both circulating fatty acids and food intake. Involvement of ROS in a model of mouse cancer cachexia

Implantation of a fast growing tumour to mice (Lewis lung carcinoma) resulted in a clear cachectic state characterized by a profound muscle wasting. This was accompanied by a significant increase in both UCP2 and UCP3 gene expression in skeletal muscle and heart. Interestingly, this increase in gene expression was not linked to a rise in circulating fatty acids or in a decrease in food intake, as previously reported in other pathophysiological states. These results question the concept that hyperlipaemia is the only factor controlling UCP gene expression in different pathophysiological conditions. In addition, the present work suggests that UCPs might participate in a counter-regulatory mechanism to lower the production of ROS.