Crystal Structure of Saccharomyces cerevisiae ECM4, a Xi-Class Glutathione Transferase that Reacts with Glutathionyl-(hydro)quinones

International audienceGlutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosyn-thesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging. The work here reports its crystallographic study. In addition to exhibiting the general GSTX structural features, ScECM4 shows extensions including a huge loop which contributes to the quaternary assembly. These structural extensions are probably specific to Saccharomycetaceae. Soaking of ScECM4 crystals with GS-menadione results in a structure where glutathione forms a mixed disulfide bond with the cysteine 46. Solution studies confirm that ScECM4 has reductase activity for GS-menadione in presence of glutathione. Moreover, the high resolution structures allowed us to propose new roles of conserved residues of the active site to assist the cysteine 46 during the catalytic act

Antioksidativnost kazeina devinog mlijeka prije i nakon in vitro simulirane enzimske razgradnje

The effect of a successive in vitro hydrolysis by pepsin and pancreatin on the free radical scavenging activity of camel milk casein was investigated in order to assess the effect of gastro-intestinal digestion. Hydrolysis of camel casein was controlled by reversed-phase high performance liquid chromatography. Anti-oxidant activity was measured by the 2,2’-azino-bis-(3-ethylbensothiazoline-6- sulfonic acid) (ABTS) method. The Trolox equivalent antioxidant capacity (TEAC) values of camel casein and its hydrolysate were 1.6±0.12 μmol TE/mg protein and 0.25 μmol TE/μmol eq. NH2, respectively. After digestion, the scavenging activity of the casein peptides was more efficient than those reported in the literature regarding digestive hydrolysates of camel milk, colostrum and whey proteins.U radu je istražen utjecaj in vitro hidrolize kazeina devinog mlijeka sa pepsinom i pankreatinom na koncentraciju slobodnih radikala i antioksidativnu aktivnost s ciljem procjenjivanja utjecaja gastro-intestinalne razgradnje. Hidroliza kazeina praćena je sa RP-HPLC (tekućinska kromatografija visoke djelotvornosti na obrnutim fazama). Antioksidativna aktivnost je praćena sa 2,2’-azino-bis-(3-ethylbensothiazoline-6-sulfonic acid) (ABTS) metodom. Ekvivalent troloks antioksidativnog kapaciteta (TEAC) devinog kazeina iznosio je 1.6±0.12 μmol TE/mg proteina, dok je TEAC hidrolizata iznosio 0.25 μmol TE/μmol eq. NH2. Nakon digestije, utvrđena antioksidativna aktivnost kazeinskih peptida bila je učinkovitija od literaturnih navoda za hidrolizate devinog mlijeka, kolostruma i sirutkinih proteina

Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3 ′ UTR

International audienceThe central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3 ′-untranslated regions (3 ′ UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3 ′ UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3 ′ UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus

EPIdemiology of Surgery-Associated Acute Kidney Injury (EPIS-AKI) : Study protocol for a multicentre, observational trial

More than 300 million surgical procedures are performed each year. Acute kidney injury (AKI) is a common complication after major surgery and is associated with adverse short-term and long-term outcomes. However, there is a large variation in the incidence of reported AKI rates. The establishment of an accurate epidemiology of surgery-associated AKI is important for healthcare policy, quality initiatives, clinical trials, as well as for improving guidelines. The objective of the Epidemiology of Surgery-associated Acute Kidney Injury (EPIS-AKI) trial is to prospectively evaluate the epidemiology of AKI after major surgery using the latest Kidney Disease: Improving Global Outcomes (KDIGO) consensus definition of AKI. EPIS-AKI is an international prospective, observational, multicentre cohort study including 10 000 patients undergoing major surgery who are subsequently admitted to the ICU or a similar high dependency unit. The primary endpoint is the incidence of AKI within 72 hours after surgery according to the KDIGO criteria. Secondary endpoints include use of renal replacement therapy (RRT), mortality during ICU and hospital stay, length of ICU and hospital stay and major adverse kidney events (combined endpoint consisting of persistent renal dysfunction, RRT and mortality) at day 90. Further, we will evaluate preoperative and intraoperative risk factors affecting the incidence of postoperative AKI. In an add-on analysis, we will assess urinary biomarkers for early detection of AKI. EPIS-AKI has been approved by the leading Ethics Committee of the Medical Council North Rhine-Westphalia, of the Westphalian Wilhelms-University Münster and the corresponding Ethics Committee at each participating site. Results will be disseminated widely and published in peer-reviewed journals, presented at conferences and used to design further AKI-related trials. Trial registration number NCT04165369

Le composant-3 des protéose-peptones du lait bovin : obtention, origine, étude de sa partie glycannique, rôle dans la lipolyse

Non disponible / Not availableLes protéose-peptones préparées par thermo coagulation du lait sont séparées par fplc d'interactions hydrophobes. Les fractions obtenues sont alors caractérisées par électrophorèse bidimensionnelle. Le composant-3, concentré dans la fraction hydrophobe est constitué par agrégation de trois sous-unités glycoprotéiques de 11, 19, et 29 kilodaltons environ. L'étude de la partie glycannique révèle que le composant-3 est n-glycolyse. La structure du n-glycanne est de type lactosaminique complexe. Le fucosyl-lacto-n-tétraose a été également identifié dans la fraction hydrophobe. Il semble présenter une forte affinité pour le composant-3. L'origine de ce dernier est liée aux protéines des membranes de globules gras du lait. Les sous-unités du composant-3 pourraient être des fragments de n-glycosylproteines membranaires obtenus par action de la plasmine à la surface des globules gras. Le mécanisme de l'inhibition de la lipolyse par le composant-3 est étudié dans un système modèle émulsifié. Il est montré que l'inhibition n'est pas due à une interaction directe entre la lipase et le composant-3, mais résulte d'un changement de la qualité de l'interface huile/eau de l'émulsion après adsorption du composant-3

Le composant-3 des proteose-peptones du lait bovin: obtention, origine etude de sa partie glycannique, role dans la lipolyse

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 83626 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Study of spontaneous deamidation of camel's milk α-lactalbumin

International audienceMany proteins are susceptible, both in vivo and in vitro, to non enzymatic deamidation that can lead to alterations in their structure and biological functions. Aspartyl and isoaspartyl residues are the primary products of this non-enzymatic reaction that proceeds through the succinimide pathway with a metastable cyclic imide as an intermediate. Camel's milk α-lactalbumin was purified by FPLC on ion exchange column (Mono Q) and the pure fraction of the α-lactalbumin was dialysed and freeze-dried. Purified α-lactalbumin was incubated for 80h at 37°C in deamidation buffer (150 mM phosphate buffer, pH 7.4 or 8.4). Protein concentration was 15 mg mL-1. At specific time points, 150 µL aliquots were withdrawn and the kinetics of spontaneous deamidation of the α-lactalbumin was followed by FPLC which shows the presence of two isoformes, this result was confirmed by electrophoresis analysis on Alkaline-PAGE. The pHi of the two isoformes was estimated by a two-dimensional isoelectrophoresis. To compare the rate of camel α-lactalbumin deamidation, the half-life of native α-lactalbumin was calculated at pH 7.4 and 8.4, the result shows that the deamidation of camel α-lactalbumin is faster at elevated pH

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein

beta-Casein was isolated from Haflinger mare’s milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine b-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare’s b-casein (25 514 6 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27–34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare’s milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30.Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif 135Asn-Gly136. Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions