Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca

<p>Abstract</p> <p>Background</p> <p>Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes.</p> <p>Results</p> <p>A marine stingray phospholipase A₂(SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl₂using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry.</p> <p>Conclusions</p> <p>SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.</p

Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

<p>Abstract</p> <p>Background</p> <p>The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC₄in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast <it>Pichia pastoris </it>in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.</p> <p>Results</p> <p>The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on <it>Pichia </it>genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.</p> <p>Conclusions</p> <p>Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.</p

Biochemical properties of pancreatic colipase from the common stingray Dasyatis pastinaca

Pancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase. In this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones.The fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.Persona

Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

<p>Abstract</p> <p>Background</p> <p>Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds.</p> <p>Results</p> <p>Chicken intestinal group IIA phospholipase A₂(ChPLA₂-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl_2,a specific activity of 160 U.mg^-1for purified ChPLA₂-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA₂-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A₂. The gene encoding the mature ChPLA₂-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA₂-IIA was built using the human intestinal phospholipase A₂structure as template.</p> <p>Conclusion</p> <p>ChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA₂-IIA.</p

Anti-inflammatory and antioxidant properties of Eriobotrya japonica leaves extracts

Background: In the present work we determined phenolic and flavonoids content of Eriobotrya japonica leaves extracts and fractions and their antioxidant and anti-inflammatory properties. Objectives: To evaluate the inhibition of inflammatory PLA2 and antioxidant effects of extracts and fractions from Eriobotrya japonica leaves Methods: Antioxidant activity was evaluated with DPPH radical scavenging assay and anti-inflammatory effect of fractions was measured by their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA). Results: The EtOH/EtOAc 2:1 extract exhibited a potent inhibition of the hG-IIA with an IC50 values of 8 \u3bcg/ml. It also shows an antioxidant activity measured on DPPH with an IC50 of 42 \u3bcg/ml. Fractionation shows that CH2Cl2/MeOH 0:1 fraction was the rich one on flavonoids compounds (4.3 mg/g dry weight) and demonstrates a high antioxidant activity with an IC50 of 12 \u3bcg/ml. The anti-inflammatory evaluation demonstrates that the same fraction was the best one to inhibit the pro-inflammatory phospholipase A2 group IIA with an IC50 of 4 \u3bcg/ml. Conclusion: Study conducted on Eriobotrya japonica shows that CH2Cl2/MeOH 0:1 fraction inhibits efficiently the hGIIA phospholipase.which is considered as pro-inflammatory enzyme

In vitro study of the PLA2 inhibition and antioxidant activities of Aloe vera leaf skin extracts

<p>Abstract</p> <p>Background</p> <p>In the present work we determined the total phenolic content of <it>Aloe vera </it>leaf skin (AVLS) extracts by using various solvents (hexane, chloroform-ethanol (1/1), ethyl acetate, butanol and water). We have also evaluated the antioxidant and the anti-PLA2 properties of these extracts by measuring their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA).</p> <p>Results</p> <p>The water extract exhibits the highest inhibitory effect with an IC₅₀= 0.22 mg/ml and interestingly no effect was observed on the digestive phospholipase A2 (group IB) even at a concentration of 5 mg/ml. Antioxidant activities were also analyzed and the most active extracts were observed when using chloroform ethanol (1/1) and ethyl acetate (IC₅₀= 0.274 and 0.326 mg/ml, respectively). Analysis of the total phenolic content reveals that the water extract, with the best anti-PLA2 effect, was poor in phenolic molecules (2 mg GAE/g). This latter value has to be compared with the chloroform-ethanol and the ethyl acetate extracts (40 and 23.8 mg GAE/g, respectively), mostly responsible for the antioxidant activity.</p> <p>Conclusion</p> <p>A significant correlation was established between the total phenolic content and the antioxidant capacity but not with the anti PLA2 activity. Results from phytochemical screening suggest that the anti PLA2 molecules were probably catechin tannins compounds.</p

Fuzzy logic-based vehicle safety estimation using V2V communications and on-board embedded ROS-based architecture for safe traffic management system in hail city

Estimating the state of surrounding vehicles is crucial to either prevent or avoid collisions with other road users. However, due to insufficient historical data and the unpredictability of future driving tactics, estimating the safety status is a difficult undertaking. To address this problem, an intelligent and autonomous traffic management system based on V2V technology is proposed. The main contribution of this work is to design a new system that uses a real-time control system and a fuzzy logic algorithm to estimate safety. The robot operating system (ROS) is the foundation of the control architechture, which connects all the various system nodes and generates the decision in the form of a speech and graphical message. The safe path is determined by a safety evaluation system that combines sensor data with a fuzzy classifier. Moreover, the suitable information processed by each vehicle unit is shared in the group to avoid unexpected problems related to speed, sudden braking, unplanned deviation, street holes, road bumps, and any kind of street issues. The connection is provided through a network based on the ZigBee protocol. The results of vehicle tests show that the proposed method provides a more reliable estimate of safety as compared to other methods

Hydatid Cyst of the Adrenal Gland: A Clinical Study of Six Cases

Hydatid cyst of the adrenal gland (HCAG) is an exceptional occurrence. We report our experience of six cases of HCAG and discuss the diagnosis and treatment of this hydatid localization. We retrospectively reviewed and analyzed the clinical files of six patients admitted to our institution from January 1990 to December 2000 for HCAG. Patients varied in age from 24—59 years. They were five males and one female. One patient had a history of pulmonary hydatidosis treated surgically 10 years previously. Five patients presented with lumbar pain and one patient had bouts of hypertension, headache, and palpitation. Physical examination was normal except in one patient who was hypertensive. Preoperative diagnosis was highly suggested by ultrasonography. CT scan performed in all cases clearly showed the relationship of the cyst with adjacent organs. Serology tests were positive in two cases. One patient had elevated urine VMA and was operated on with the diagnosis of cystic phaeochromocytoma. All six patients were operated on and had either an adrenalectomy (two cases) or partial pericystectomy (four cases). In one case, partial pericystectomy was conducted through a retroperitoneal laparoscopic approach. The hydatid nature of the cyst was confirmed pathologically. All patients had a smooth postoperative course with no cystic recurrence on follow-up. The diagnosis of HCAG is based mainly on ultrasonography and CT scan. Surgery with either partial or total excision of the cyst, with or without preservation of the adrenal gland, is the treatment of choice

SARS-CoV-2 seroprevalence in the urban population of Qatar: An analysis of antibody testing on a sample of 112,941 individuals

ABSTRACTBackgroundQatar has experienced a large SARS-CoV-2 epidemic. Our first objective was to assess the proportion of the urban population that has been infected with SARS-CoV-2, by measuring the prevalence of detectable antibodies. Our second objective was to identify predictors for infection and for having higher antibody titers.MethodsResidual blood specimens from individuals receiving routine and other clinical care between May 12-September 9, 2020 were tested for anti-SARS-CoV-2 antibodies. Associations with seropositivity and higher antibody titers were identified through regression analyses. Probability weights were applied in deriving the epidemiological measures.ResultsWe tested 112,941 individuals (∼10% of Qatar’s urban population), of whom 51.6% were men and 66.0% were 20-49 years of age. Seropositivity was 13.3% (95% CI: 13.1-13.6%) and was significantly associated with sex, age, nationality, clinical-care type, and testing date. The proportion with higher antibody titers varied by age, nationality, clinical-care type, and testing date. There was a strong correlation between higher antibody titers and seroprevalence in each nationality, with a Pearson correlation coefficient of 0.85 (95% CI: 0.47-0.96), suggesting that higher antibody titers may indicate repeated exposure to the virus. The percentage of antibody-positive persons with prior PCR-confirmed diagnosis was 47.1% (95% CI: 46.1-48.2%), severity rate was 3.9% (95% CI: 3.7-4.2%), criticality rate was 1.3% (95% CI: 1.1-1.4%), and fatality rate was 0.3% (95% CI: 0.2-0.3%).ConclusionsFewer than two in every 10 individuals in Qatar’s urban population had detectable antibodies against SARS-CoV-2 between May 12-September 9, 2020, suggesting that this population is still far from the herd immunity threshold and at risk from a subsequent epidemic wave.</jats:sec