Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.     

Are Phosphatidylcholine and Lysophosphatidylcholine Body Levels Potentially Reliable Biomarkers in Obesity? A Review of Human Studies

Phosphatidylcholines (PCs) are the major components of biological membranes in animals and are a class of phospholipids that incorporate choline as a headgroup. Lysophosphatidylcholines (LPCs) are a class of lipid biomolecules derived from the cleavage of PCs, and are the main components of oxidized low-density lipoproteins (oxLDLs) that are involved in the pathogenesis of atherosclerosis. Since obesity is associated with a state of chronic low-grade inflammation, one can anticipate that the lipidomic profile changes in this context and both PCs and LPCs are gaining attention as hypothetically reliable biomarkers of obesity. Thus, a literature search is performed on PubMed, Latin American and Caribbean Health Science Literature (LILACS), and Excerpta Medica DataBASE (Embase) to obtain the findings of population studies to clarify this hypothesis. The search strategy resulted in a total of 2403 reports and 21 studies were included according to the eligibility criteria. Controversial data on the associations of PCs and LPCs with body mass index (BMI) and body fat parameters have been identified. There is an inverse relationship between BMI and most species of PCs, and a majority of studies exhibited negative associations between BMI and LPCs. Other findings regarding the differences between PCs and LPCs in obesity are presented, and the associated uncertainties are discussed in detail.     

Update on optimized purification and characterization of natural milk allergens

Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.     

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.     

Correlation between airborne particle concentrations in seven industrial plants and estimated respiratory tract deposition by number,mass and elemental composition

The number and mass distribution of airborne particles were recorded in several industrial plants. From the data obtained, particle deposition was estimated in three regions of the respiratory tract using the ICRP grand average deposition model based on Hinds' (1999) parameterization. The median diameter was 30–70 nm (number distributions), and >4 μm (mass distributions) near most work activities, resulting in linear relationships between the deposited number/mass concentrations and the number/mass concentrations in the air. Welding and laser cutting produced particles in the 200–500-nm range; total deposition was small, not in accordance with the linear relationship observed for the other work activities. The elemental content varied between particle sizes in some workplaces, causing different elements to deposit in different respiratory regions. Iron was the most abundant element in the particles in many of the workplaces; in an iron foundry, however, Fe was most abundant only in the micron-sized particles whereas the nanoparticles mainly comprised Pb and Sb.     

Thioether Analogues of Disulfide‐Bridged Cyclic Peptides Targeting Death Receptor 5: Conformational Analysis,Dimerisation and Consequences for Receptor Activation

Cyclic peptides containing redox‐stable thioether bridges might provide a useful alternative to disulfide‐bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16‐mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL‐R2). Upon covalent oligomerisation, the disulfide‐bridged peptide has previously shown similar behaviour to that of TNF‐related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5‐binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5‐mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.     

Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity

Substrate‐promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio‐ and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)‐lisofylline, a compound under investigation for its anti‐inflammatory properties, was chosen as a first proof‐of‐principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.     

Thermal Ageing Induced Effects on Pd/LaFeO3 for NOx Reduction by Hydrocarbons: Influence of the Preparation Method

The influence of the support synthesis for the preparation of supported Pd/LaFeO₃ perovskite based catalysts and the nature of the palladium precursor on the catalytic performances in the selective reduction of NO to N₂ by hydrocarbons have been investigated. According to the preparation method, subsequent surface and bulk characterisation revealed significant changes in the degree of dispersion and the chemical environment of oxidic palladium species depending on the extent of interaction with the support. Pd nitrate precursor leads to the stabilisation of the Pd surface concentration. The metal support interactions thus obtained strongly enhance the catalytic performances, particularly the resistance to thermal ageing whereas sol–gel synthesis produces the higher support activity.