Louis Sarda

Bio: Louis Sarda is an academic researcher from University of Provence. The author has contributed to research in topics: Colipase & Lipase. The author has an hindex of 33, co-authored 73 publications receiving 4382 citations. Previous affiliations of Louis Sarda include Centre national de la recherche scientifique.

Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells

Abstract: Nonpancreatic secretory phospholipase A 2 (sPLA 2 ) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA 2 hydrolyzed phospholipids in membrane microvesicles shed from Ca 2+ -loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA 2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA 2 were detected in inflammatory fluids in relative proportions identical to those used in vitro. We conclude that upon loss of phospholipid asymmetry, cell-derived microvesicles provide a preferential substrate for sPLA 2 . SPH hydrolysis, which is provoked by various cytokines, regulates sPLA 2 activity, and the novel lipid mediator LPA can be generated by this pathway.

Structure of the pancreatic lipase-procolipase complex

Abstract: Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit.

Abstract: We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.

Purification from porcine pancreas of two molecular species with lipase activity

Abstract: The two lipases existing in porcine pancreas and pancreatic juice have been fully purified by a method involving the six following steps: (1) almost complete delipidation of pancreas homogenates by solvent extraction; (2) (NH4)2SO4 fractionation; (3) removal of an acidic phosphatide by extraction and partition between n butanol and (NH4)2SO4; (4) chromatography on DEAE-cellulose (pH 9.0); (5) filtration through Sephadex G-100 and (6) separation of the two lipases by chromatography on CM-cellulose (pH 5.0). This method can be applied on a relatively large preparative scale. The two porcine lipases appear to be very closely related with regard to amino acid composition, molecular weight (45 000–50 000) and specific activity on long- or short-chain triglycerides.

Kinetic Assay of Human Gastric Lipase on Short and Long Chain Triacylglycerol Emulsions

Abstract: The hydrolysis of dietary triacylglycerol in human begins in the stomach and is catalysed by acid stable lipases present in gastric juice (1–10).

A standardised static in vitro digestion method suitable for food – an international consensus

Abstract: Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Abstract: The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

Industrial applications of microbial lipases

Abstract: Lipases are a class of enzymes which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases constitute the most important group of biocatalysts for biotechnological applications. This review describes various industrial applications of microbial lipases in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery.

Cytokines in atherosclerosis: pathogenic and regulatory pathways.

Abstract: Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immunoinflammatory mechanisms are involved. Inflammation is central at all stages of atherosclerosis. It is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines and adhesion molecules leading to monocyte/lymphocyte recruitment and infiltration into the subendothelium. It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading to rupture and thrombus formation. Cells involved in the atherosclerotic process secrete and are activated by soluble factors, known as cytokines. Important recent advances in the comprehension of the mechanisms of atherosclerosis provided evidence that the immunoinflammatory response in atherosclerosis is modulated by regulatory pathways, in which the two anti-inflammatory cytokines interleukin-10 and transforming growth factor-beta play a critical role. The purpose of this review is to bring together the current information concerning the role of cytokines in the development, progression, and complications of atherosclerosis. Specific emphasis is placed on the contribution of pro- and anti-inflammatory cytokines to pathogenic (innate and adaptive) and regulatory immunity in the context of atherosclerosis. Based on our current knowledge of the role of cytokines in atherosclerosis, we propose some novel therapeutic strategies to combat this disease. In addition, we discuss the potential of circulating cytokine levels as biomarkers of coronary artery disease.

Production, purification, characterization, and applications of lipases.

Abstract: Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing.