Yvan Choiset

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants.

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR). To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag.

Aims:  The aim of this study was to isolate and identify bacteriocin‐producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mares milk), and to purify and characterize bacteriocins produced by these LAB.

Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3.

New proteinaceous active substance produced by Lactobacillus paracasei subsp. paracasei strain M3 used as a starter for Bulgarian yellow cheese was identified and studied. It displayed bactericidal and fungistatic activities. Its activity was checked against over 60 bacterial and yeast strains. It was efficient against Bacillus subtilis ATCC 6633, several L. delbrueckii species, Helicobacter pylori NCIPD 230 and some yeast species, for example Candida albicans, C. pseudointermedia NBIMCC 1532, C. blankii NBIMCC 85 and Saccharomyces cerevisiae NBIMCC 1812. The synthesis of the substance by producing strain was detected in the late logarithmic growth phase during batch fermentation. Anion exchange chromatography, reversed phase chromatography (RPC) on C4 column and HPLC on C18 column were used for partial purification of this antimicrobial compound. The gene responsible for the synthesis of the active substance is located on the bacterial chromosome.

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C(18) column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing.

Glycodelin and β‐lactoglobulin, lipocalins with a high structural similarity, differ in ligand binding properties

Human glycodelin, a lipocalin with a high amino acid similarity to β‐lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin‐A) and seminal plasma (glycodelin‐S). We found that the structures of these glycodelins and β‐lactoglobulin are similar. Despite this structural similarity, unlike β‐lactoglobulin, glycodelin‐A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin‐A and glycodelin‐S is not influenced by the differences in glycosylation or by ligand binding.

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41-K60; Y102-R124; L149-I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS-PAGE and RP-HPLC. Peptides released were identified by LC-MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3-5year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.

Interactions of β-Lactoglobulin Variants A and B with Vitamin A. Competitive Binding of Retinoids and Carotenoids

β-Lactoglobulin (β-Lg) is the major whey protein of bovine milk present at a concentration of 2-3 g L(-1). Its biological role is still not well-known. However, many studies have suggested that β-Lg may play either nutritional or specific transporter role. The high affinity of β-Lg for retinol and other retinoids was reported. The results of interaction studies of β-Lg with carotenoids, that is, β-carotene, β-cryptoxanthin, and α-carotene, which display similar structures are reported in this study. The affinities of β-Lg for binding of retinoids and carotenoids were compared, providing more information about the binding site(s) of these molecules by β-Lg. Interactions were followed by the measurements of quenching of β-Lg tryptophan fluorescence and retinol fluorescence. The obtained results indicate that carotenoids are bound by β-Lg with high affinity of the order of 10(-8) M. Measurement of retinol competition with carotenoids for binding by β-Lg suggests that the binding of these two ligands occurs at two different sites of β-Lg.

Antimicrobial and antifungal activities of Lactobacillus curvatus strain isolated from homemade Azerbaijani cheese.

The aims of this study were to characterize inhibitory activity spectra, some probiotic properties and safety of Lactobacillus curvatus A61 for its future application in production of fermented foods. The studied strain was isolated from traditional homemade cheese manufactured in Azerbaijan. The cell-free supernatant of culture of Lb. curvatus A61 inhibited the growth of tested LAB, as well as of Listeria monocytogenes and Bacillus cereus strains. The strain presented antifungal activity and inhibited the growth of Cladosporium and Fusarium ssp. during co-cultivation on agar media. PCR amplification with specific primers revealed the presence of curvacin A encoding gene in Lb. curvatus A61. Bacteriocin produced by the studied strain was heat stable and active in a broad pH range, and in the presence of Triton X-20, Triton X-80, Triton X-100, β-mercaptoethanol, Na-EDTA, SDS and NaCl. The mode of action of bacteriocin against selected indicator strains was found to be bacteriostatic. Lb. curvatus A61 was resistant to physiological concentrations of bile salts and showed high auto-aggregation ability, as well as co-aggregation ability with pathogenic L. monocytogenes strains. It was sensitive to chloramphenicol, penicillin, tetracycline, ciprofloxacin and vancomycin, but resistant to ampicillin and gentamicin.

Proteolysis by Lactobacillus fermentum IFO3956 isolated from Egyptian milk products decreases immuno-reactivity of αS1-casein.

Proteinase activity of Lactobacillus fermentum IFO3956 cells was higher when they were grown on milk-based media than on 10% reconstituted skim milk. The lowest protease activity was observed when cells were grown on milk-free media. The extraction of milk-induced cell-bound proteases from Lb. fermentum IFO3956 was most efficient using 1% Tween 80 while the use of 1% SDS inhibited all proteolytic activity. Two bands of ∼35 and >100 kDa were observed by zymogram, indicating that proteolytic activity corresponded to the presence of at least two types of enzymes or two molecular forms of one enzyme. Mass spectrometry analyses of αS1-casein hydrolysates detected 24 peptides with sizes ranging from 5 to 36 amino acids, including 9 phosphorylated peptides, resulting from the fermentation of Lb. fermentum IFO3956 of αS1-casein. Most of the identified peptides originated from the N-terminal portion of αS1-casein. The studied bacterial strain could hydrolyze αS1-casein in many sites including the epitopes triggering the allergic reactions against αS1-casein e.g. at the positions 23, 30, 41, 71, 91, 98, 126, 179. After hydrolysis of αS1-casein with Lb. fermentum IFO3956 the recognition and the binding of this casein to IgE from the pooled sera of 18 patients with cows milk allergy was significantly reduced.

Interactions of β-lactoglobulin with serotonin and arachidonyl serotonin.

β-Lactoglobulin (β-LG) is a lipocalin, which is the major whey protein of cows milk and the milk of other mammals. However, it is absent from human milk. The biological function of β-LG is not clear, but its potential role in carrying fatty acids through the digestive tract has been suggested. β-LG has been found in complexes with lipids such as butyric and oleic acids and has a high affinity for a wide variety of compounds. Serotonin (5-hydroxytryptamine, 5-HT), an important compound found in animals and plants, has various functions, including the regulation of mood, appetite, sleep, muscle contraction, and some cognitive functions such as memory and learning. In this study, the interaction of serotonin and one of its derivatives, arachidonyl serotonin (AA-5HT), with β-LG was investigated using circular dichroism (CD) and fluorescence intensity measurements. These two ligands interact with β-LG forming equimolar complexes. The binding constant for the serotonin/β-LG interaction is between 10⁵ and 10⁶ M(-1) , whereas for the AA-5HT/β-LG complex it is between 10⁴ and 10⁵ M(-1) as determined by measurements of either protein or ligand fluorescence. The observed binding affinities were higher in hydroethanolic media (25% EtOH). The interactions between serotonin/β-LG and AA-5HT/β-LG may compete with self-association (micellization) of both the ligand and the protein. According to far- and near-UV CD results, these ligands have no apparent influence on β-LG secondary structure, however they partially destabilize its tertiary structure. Their binding by β-LG may be one of the peripheral mechanisms of the regulation of the content of serotonin and its derivatives in the bowel of milk-fed animals.