Proteolytic cleavage of stingray phospholipase A2: Isolation and biochemical characterization of an active N-terminal form

<p>Abstract</p> <p>Background</p> <p>Mammalian GIB-PLA2 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. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2).</p> <p>Results</p> <p>The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze SPLA2 in different ways. In both cases, only N-terminal fragments were accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic and chymotryptic attack generated 13 kDa and 12 kDa forms of SPLA2, respectively. Interestingly, the SPLA2 13 kDa form was inactive, whereas the SPLA2 12 kDa form conserved almost its full phospholipase activity. In the absence of bile slats both native and 12kDa SPLA2 failed to catalyse the hydrolysis of PC emulsion. When bile salts were pre-incubated with the substrate, the native kinetic protein remained linear for more than 25 min, whereas the 12 kDa form activity was found to decrease rapidly. Furthermore, The SPLA2 activity was dependent on Ca²⁺; other cations (Mg²⁺, Mn²⁺, Cd²⁺and Zn²⁺) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca²⁺.</p> <p>Conclusions</p> <p>Although marine and mammal pancreatic PLA2 share a high amino acid sequence homology, polyclonal antibodies directed against SPLA2 failed to recognize mammal PLA2 like the dromedary pancreatic one. Further investigations are needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of phospholipases.</p

Etiology of autistic features: the persisting neurotoxic effects of propionic acid

BACKGROUND: Recent clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism. Propionic acid (PA) is a short chain fatty acid and an important intermediate of cellular metabolism. Although PA has several beneficial biological effects, its accumulation is neurotoxic. METHODS: Two groups of young Western albino male rats weighing about 45 to 60 grams (approximately 21 days old) were used in the present study. The first group consisted of oral buffered PA-treated rats that were given a neurotoxic dose of 250 mg/kg body weight/day for three days, n = eight; the second group of rats were given only phosphate buffered saline and used as a control. Biochemical parameters representing oxidative stress, energy metabolism, neuroinflammation, neurotransmission, and apoptosis were investigated in brain homogenates of both groups. RESULTS: Biochemical analyses of brain homogenates from PA-treated rats showed an increase in oxidative stress markers (for example, lipid peroxidation), coupled with a decrease in glutathione (GSH) and glutathione peroxidase (GPX) and catalase activities. Impaired energy metabolism was ascertained through the decrease of lactate dehydrogenase and activation of creatine kinase (CK). Elevated IL-6, TNFα, IFNγ and heat shock protein 70 (HSP70) confirmed the neuroinflammatory effect of PA. Moreover, elevation of caspase3 and DNA fragmentation proved the pro-apoptotic and neurotoxic effect of PA to rat pups CONCLUSION: By comparing the results obtained with those from animal models of autism or with clinical data on the biochemical profile of autistic patients, this study showed that the neurotoxicity of PA as an environmental factor could play a central role in the etiology of autistic biochemical features

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

Proinflammatory and proapoptotic markers in relation to mono and di-cations in plasma of autistic patients from Saudi Arabia

<p>Abstract</p> <p>Objectives</p> <p>Autism is a developmental disorder characterized by social and emotional deficits, language impairments and stereotyped behaviors that manifest in early postnatal life. This study aims to clarify the relationship amongst absolute and relative concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺and/or proinflammatory and proapoptotic biomarkers.</p> <p>Materials and methods</p> <p>Na⁺, K⁺, Ca²⁺, Mg²⁺, Na⁺/K⁺, Ca²⁺/Mg²⁺together with IL6, TNFα as proinflammatory cytokines and caspase3 as proapoptotic biomarker were determined in plasma of 25 Saudi autistic male patients and compared to 16 age and gender matching control samples.</p> <p>Results</p> <p>The obtained data recorded that Saudi autistic patients have a remarkable lower plasma caspase3, IL6, TNFα, Ca²⁺and a significantly higher K⁺compared to age and gender matching controls. On the other hand both Mg²⁺and Na⁺were non-significantly altered in autistic patients. Pearson correlations revealed that plasma concentrations of the measured cytokines and caspase-3 were positively correlated with Ca²⁺and Ca²⁺/K⁺ratio. Reciever Operating Characteristics (ROC) analysis proved that the measured parameters recorded satisfactory levels of specificity and sensitivity.</p> <p>Conclusion</p> <p>Alteration of the selected measured ions confirms that oxidative stress and defective mitochondrial energy production could be contributed in the pathogenesis of autism. Moreover, it highlights the relationship between the measured ions, IL6, TNFα and caspase3 as a set of signalling pathways that might have a role in generating this increasingly prevalent disorder. The role of ions in the possible proinflammation and proapoptic mechanisms of autistics' brains were hypothesized and explained.</p

Plasma fatty acids as diagnostic markers in autistic patients from Saudi Arabia

<p>Abstract</p> <p>Backgrounds</p> <p>Autism is a family of developmental disorders of unknown origin. The disorder is characterized by behavioral, developmental, neuropathological and sensory abnormalities, and is usually diagnosed between the ages of 2 and 10 with peak prevalence rates observed in children aged 5-8 years. Recently, there has been heightened interest in the role of plasma free fatty acids (FA) in the pathology of neurological disorders. The aim of this study is to compare plasma fatty acid profiles of Saudi autistic patients with those of age-matching control subjects in an attempt to clarify the role of FA in the etiology of autism.</p> <p>Methods</p> <p>26 autistic patients together with 26-age-matching controls were enrolled in the present study. Methyl esters of FA were extracted with hexane, and the fatty acid composition of the extract was analyzed on a gas chromatography.</p> <p>Results</p> <p>The obtained data proved that fatty acids are altered in the plasma of autistic patients, specifically showing an increase in most of the saturated fatty acids except for propionic acid, and a decrease in most of polyunsaturated fatty acids. The altered fatty acid profile was discussed in relation to oxidative stress, mitochondrial dysfunction and the high lead (Pb) concentration previously reported in Saudi autistic patients. Statistical analysis of the obtained data shows that most of the measured fatty acids were significantly different in autistic patients compared to age -matching controls.</p> <p>Conclusions</p> <p>Receiver Operating Characteristic (ROC) curve analysis shows satisfactory values of area under the curve (AUC) which could reflect the high degree of specificity and sensitivity of the altered fatty acids as biomarkers in autistic patients from Saudi Arabia.</p

Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

AbstractAn extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60°C and pH 12 by using olive oil emulsion or TC4, reached 7215U/mg and 2484U/mg, respectively. The 38 NH2-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0–12 after a 24h incubation or after 0.5h treatment at 70°C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry

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

Synthesis and evaluation of anticancer, antiphospholipases, antiproteases, and antimetabolic syndrome activities of some 3H-quinazolin-4-one derivatives

Some new 3H-quinazolin-4-one derivatives were synthesised and screened for anticancer, antiphospholipases, antiproteases, and antimetabolic syndrome activities. Compound 15d was more potent in reducing the cell viabilities of HT-29 and SW620 cells lines to 38%, 36.7%, compared to 5-FU which demonstrated cell viabilities of 65.9 and 42.7% respectively. The IC50 values of 15d were ∼20 µg/ml. Assessment of apoptotic activity revealed that 15d decreased the cell viability by down regulating Bcl2 and BclxL. Moreover, compounds, 8j, 8d/15a/15e, 5b, and 8f displayed lowered IC50 values than oleanolic acid against proinflammatory isoforms of hGV, hG-X, NmPLA2, and AmPLA2. In addition, 8d, 8h, 8j, 15a, 15b, 15e, and 15f showed better anti-α-amylase than quercetin, whereas 8g, 8h, and 8i showed higher anti-α-glucosidase activity than allopurinol. Thus, these compounds can be considered as potential antidiabetic agents. Finally, none of the compounds showed higher antiproteases or xanthine oxidase activities than the used reference drugs

<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:"Times New Roman";mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-GB;mso-fareast-language: EN-US;mso-bidi-language:AR-SA" lang="EN-GB">Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic <i>Bacillus</i> <i>stearothermophilus</i> </span>

179-188Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55°C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55°C and retained more than 70% of its activity after incubation at 70°C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations