Chromatography analysis of seminal plasma proteins in buffalo semen samples with high and low cryotolerance.

The aim of this study was to analyse and identify specific buffalo seminal plasma proteins (SPPs) responsible for sperm cryotolerance during low temperature storage. Computer Assisted Sperm Analysis (CASA) of the motility and viability of buffalo spermatozoa was performed before freezing and after thawing. Two sample groups were formed - ejaculates with high cryotol- erance (group A) and low cryotolerance (group B). CASA demonstrated that the initial progres- sive motility after thawing of the spermatozoa in group A is significantly higher than in group B (p⟨0.001). Group B showed a significant increase in the percentage of static and non-progressive spermatozoa at 240 min, when compared to group A (p⟨0.05). SPPs, proteins in the cryoprotec- tive medium (PM) and proteins in the mixture of PM and SP were separated by High Perfor- mance Liquid Chromatography (HPLC). Comparative analysis of the chromatographic profiles was performed to identify specific proteins related to sperm cryotolerance. SPPs profiles showed 5 distinct protein peaks in both groups, ranging from 500 kDa to 50 Da. Chromatograms of group A and group B showed quantitative and qualitative differences in protein content. Chromato- grams of proteins in PM showed 11 well-expressed peaks. HPLC analysis of the mixtures of SPPs from the two groups and PM visualized the formation of a new bio-complex structure expressed by a protein peak specific for group A (7.674 min, AU 1.50). This protein peak can be referred as a phenotypic trait for buffalo ejaculates with high sperm cryotolerance.

Association of +3179G/A insulin-like growth factor-1 receptor polymorphism and insulin-like growth factor-1 serum level with systemic lupus erythematosus.

The insulin-like growth factor (IGF) system plays a prominent role in the regulation of immunity and inflammation. Inappropriate balance of IGF-1 signaling has been reported in autoimmune disorders. This study was designed to compare +3179G/A IGF-1R genotype distribution in 148 systemic lupus erythematosus (SLE) patients with a group of 240 healthy donors. We also investigated serum IGF-1 levels in SLE patients and healthy controls in an association to genotype. IGF-1 serum levels were measured by enzyme-linked immunosorbent assay and genotyping for the +3179G/A polymorphism was performed by restriction fragment length polymorphisms (RFLP)-polymerase chain reaction (PCR) assay. The higher frequency of homozygous genotype AA (22% vs. 17% with OR 1.319, 95% CI 0.71--2.44) and lower frequency of heterozygous genotype AG (42% vs. 46% with OR 0.698, 95% CI 0.38-1.27) were seen in cases versus controls. Serum IGF-1 levels were comparable between SLE patients and age- and sex-matched healthy donors, even when the groups was stratified according to +3179G/A IGF-1R genotypes. However, when patients were sub grouped according to the disease activity index (SLEDAI score), serum IGF-1 levels were increased in patients with severe disease activity. These results indicated that systemic lupus erythematosus activity is affected by a modulation of the insulin-like growth factor-1 signal pathway and +3179G/A IGF-1R polymorphism.

Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants.

Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.

A new method for determining phospholipase D activity using the monomolecular film technique.

A versatile and continuous assay for phospholipase D (PL D) activity was developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of stable 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generated a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-soluble choline. Secondly, the latter acidic phospholipid, in contrast to the initial PC molecule, was further hydrolysed under the action of porcine pancreatic lipase (PPL) in order to give rise to lysophosphatidic acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the PL D-catalyzed reaction with phospholipid monolayers as substrates. The PLD kinetics were linear with time and the velocities recorded were directly dependent upon the amount of PL D used. In a preliminary study, we investigated the effects of the surface pressure on the PL D activity.

Glyceride synthesis catalyzed by cutinase using the monomolecular film technique.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

Interactions between beta-cyclodextrin and insoluble glyceride monomolecular films at the argon/water interface: application to lipase kinetics.

A study of the desorption rate of insoluble monomolecular films of oleic acid (OA), monoolein (MO), 1,2-diolein (1,2-DO), 1,3-diolein (1,3-DO) and triolein (TO) at the argon/water interface by water-soluble beta-cyclodextrin (beta-CD) is reported. The desorption of OA and MO involves probably the complexation of the single acyl chain with beta-CD and sequestering of the formed soluble OA/beta-CD and MO/beta-CD complexes from the argon/water interface. In the case of monolayers of multiple acyl chain molecules such as DO and TO, no detectable change in the surface pressure occurred after beta-CD injection. The surface rheological dilatational properties of the monolayers of DO and TO in the presence of beta-CD in the subphase were studied. The elasticity of the DO monolayer remained unchanged, whereas the decrease in the surface elasticity of the TO film was attributed to the formation of a water-insoluble TO/beta-CD complex. With the 'tuning fork' model, one acyl chain of TO can be included in the beta-CD cavity. The formed TO/beta-CD complex present at the interface retarded the propagation of the dilatational deformation along the plane of the monolayer. Schematic models have been proposed in an attempt to explain the different complexation of these lipids by beta-CD at the argon/water interface. In addition to the above results, the presence of beta-CD in the water subphase makes it possible for the first time to perform kinetic measurements of the lipase hydrolysis rates of long-chain glycerides forming monomolecular films.

The condensing effects of egg lecithin and cholesterol on triolein monolayers are inhibited by substitution of one saturated acyl chain in the triacylglycerol.

Previous work showed that the clearance from plasma of chylomicron-like emulsions injected intravenously was affected by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing triolein (OOO) as the only triacylglycerol, clearances were decreased by a single saturated chain in emulsions containing 1,3-dioleoyl-2-stearoyl-sn-glycerol (OSO), 1,2-dioleoyl-3-stearoyl-sn-glycerol (OOS) or 1-stearoyl-2,3-dioleoyl-sn-glycerol (SOO). The differences in clearance may reflect physical differences at the oil-water interface related to chain interactions of the triacylglycerol structures with other lipid components. In the present work lipid monomolecular films at the air-water interface were used to establish the capacity of OOO to interact with the pure synthetic triacylglycerols OOS and SOO, and the capacity of OOS and SOO to co-exist in monolayers of lecithin and of cholesterol was compared with OOO. Substituting one oleoyl chain by a stearoyl chain induced a 20% condensation in monomolecular films of the pure triacylglycerols. Mixtures of OOO with either pure egg yolk phosphatidylcholine or cholesterol also showed substantial condensing effects. In contrast substituting one oleoyl chain by a stearoyl chain substantially lessened the condensing effects. At surface pressures above the collapse pressure of the pure triacylglycerols, substantially more OOO than OOS or SOO was retained in mixed monolayers with phosphatidylcholine. These differences could underlie the effects on metabolism of saturated chains in emulsion triacylglycerols.

Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies.

Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgG1 class with a kappa light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.