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

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia

<p>Abstract</p> <p>Background</p> <p>In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of <it>M. vulgare </it>L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.</p> <p>Methods</p> <p>The agar disk diffusion method was used to study the antibacterial activity of <it>M. vulgare </it>essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC₅₀) were investigated to characterize the antimicrobial activities of this essential oil. The <it>in vitro </it>cytotoxicity of <it>M. vulgare </it>essential oil was examined using a modified MTT assay; the viability and the IC₅₀were used to evaluate this test.</p> <p>Results</p> <p>The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, <it>Botrytis cinerea </it>exhibited the strongest activity with inhibition zones of 12.6 mm. However, <it>Fusarium solani, Penicillium digitatum </it>and <it>Aspergillus niger </it>were less sensitive to <it>M. vulgare </it>essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC₅₀value of 0.258 μg/ml.</p> <p>Conclusion</p> <p>This investigation showed that the <it>M. vulgare </it>essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and <it>Botrytis cinerea </it>fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.</p

Antagonistic properties of some halophilic thermoactinomycetes isolated from superficial sediment of a solar saltern and production of cyclic antimicrobial peptides by the novel isolate paludifilum halophilum

This study has focused on the isolation of twenty-three halophilic actinomycetes from two ponds of different salinity and the evaluation of their ability to exert an antimicrobial activity against both their competitors and several other pathogens. From the 23 isolates, 18 strains showed antagonistic activity, while 19 showed activities against one or more of the seven pathogen strains tested. Six strains exhibited consistent antibacterial activity against Gram-negative and Gram-positive pathogens characterized at the physiological and molecular levels. These strains shared only 94-95% 16S rRNA sequence identity with the closely related species of the Thermoactinomycetaceae family. Among them, the potent strain SMBg3 was further characterized and assigned to a new genus in the family for which the name Paludifilum halophilum (DSM 102817T) is proposed. Sequential extraction of the antimicrobial compounds with ethyl acetate revealed that the crude extract from SMBg3 strain had inhibitory effect on the growth of the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa. Based on the HRESI-MS spectral data, the cyclic lipopeptide Gramicidin S and four cyclic dipeptides (CDPs) named cyclo(L-4-OH-Pro-L-Leu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(L-Leu-L-Pro) were detected in the fermentation broth of Paludifilum halophilum. To our knowledge, this is the first report on the isolation of these compounds from members of the Thermoactinomycetaceae family

Insights into the mechanisms governing P01 scorpion toxin effect against U87 glioblastoma cells oncogenesis

The emerging concept of small conductance Ca2+-activated potassium channels (SKCa) as pharmacological target for cancer treatment has significantly increased in recent years. In this study, we isolated the P01 toxin from Androctonus australis (Aa) scorpion venom and investigated its effect on biological properties of glioblastoma U87, breast MDA-MB231 and colon adenocarcinoma LS174 cancer cell lines. Our results showed that P01 was active only on U87 glioblastoma cells. It inhibited their proliferation, adhesion and migration with IC50 values in the micromolar range. We have also shown that P01 reduced the amplitude of the currents recorded in HEK293 cells expressing SK2 channels with an IC50 value of 3 pM, while it had no effect on those expressing SK3 channels. The investigation of the SKCa channels expression pattern showed that SK2 transcripts were expressed differently in the three cancer cell lines. Particularly, we highlighted the presence of SK2 isoforms in U87 cells, which could explain and rely on the specific activity of P01 on this cell line. These experimental data highlighted the usefulness of scorpion peptides to decipher the role of SKCa channels in the tumorigenesis process, and develop potential therapeutic molecules targeting glioblastoma with high selectivity

Comparative study of kinetic and interfacial properties of a novel

We compared several kinetic and interfacial properties of a lipase from a novel strain of Rhizopus oryzae (ROLw) with ROL29 lipase. In contrast to ROL29, ROLw was able to hydrolyze triolein emulsion in the absence of any additive, like bovine serum albumin (BSA). Furthermore, unlike Rhizopus oryzae lipase (ROL29), kinetic study of ROLw lipase shows linear dependency when using tributyrin emulsion as substrate. ROLw can tolerate, more efficiently than ROL29, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate. The critical surface pressure πc of penetration into phosphatidyl choline from egg yolk films was found to be 23 mN/m with ROLw, in contrast to a value of 10 mN/m obtained with ROL29. The effect of calcium ion and synthetic detergent on the two lipases was studied. In contrast to ROL29, ROLw was activated in the presence of 100 lmoles TX-100. No significant difference on the two lipase activity was observed in presence or absence of calcium ion

Comparative study of kinetic and interfacial properties of a novel Rhizopus oryzae lipase and ROL29

We compared several kinetic and interfacial properties of a lipase from a novel strain of Rhizopus oryzae (ROLw) with ROL29 lipase. In contrast to ROL29, ROLw was able to hydrolyze triolein emulsion in the absence of any additive, like bovine serum albumin (BSA). Furthermore, unlike Rhizopus oryzae lipase (ROL29), kinetic study of ROLw lipase shows linear dependency when using tributyrin emulsion as substrate. ROLw can tolerate, more efficiently than ROL29, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate. The critical surface pressure πc of penetration into phosphatidyl choline from egg yolk films was found to be 23 mN/m with ROLw, in contrast to a value of 10 mN/m obtained with ROL29. The effect of calcium ion and synthetic detergent on the two lipases was studied. In contrast to ROL29, ROLw was activated in the presence of 100 lmoles TX-100. No significant difference on the two lipase activity was observed in presence or absence of calcium ion

Comparative study of kinetic and interfacial properties of a novel Rhizopus oryzae

We compared several kinetic and interfacial properties of a lipase from a novel strain of Rhizopus oryzae (ROLw) with ROL29 lipase. In contrast to ROL29, ROLw was able to hydrolyze triolein emulsion in the absence of any additive, like bovine serum albumin (BSA). Furthermore, unlike Rhizopus oryzae lipase (ROL29), kinetic study of ROLw lipase shows linear dependency when using tributyrin emulsion as substrate. ROLw can tolerate, more efficiently than ROL29, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate. The critical surface pressure πc of penetration into phosphatidyl choline from egg yolk films was found to be 23 mN/m with ROLw, in contrast to a value of 10 mN/m obtained with ROL29. The effect of calcium ion and synthetic detergent on the two lipases was studied. In contrast to ROL29, ROLw was activated in the presence of 100 lmoles TX-100. No significant difference on the two lipase activity was observed in presence or absence of calcium ion

<span style="font-size:15.0pt;mso-bidi-font-weight:bold" lang="EN-US">Purification and characterization of a phospholipase A₂-IIA from common stingray (<i>Dasyatis pastinaca)</i> intestine </span>

186-195<span style="font-size:9.0pt;mso-bidi-font-size: 11.0pt" lang="EN-US">A phospholipase A2 belonging to IIA group secretory PLA2 was isolated and purified to homogeneity from the intestine of common stingray (Dasyatis pastinaca) using acidic treatment (pH 1.5) and ammonium sulphate precipitation methods combined with single-column ion-exchange chromatography. The purified enzyme was found to be a glycosylated monomeric protein with a molecular mass of about 14 kDa. The stingray sPLA2-IIA had optimum activity at 45°C, unlike known mammalian PLA2-IIAs, which show optimum activity at 37°C. The purified enzyme exhibited a specific activity of 290 U/mg at optimal conditions (pH 9.5 and 45°C) in the presence of 6 mM NaDC and 8 mM CaCl2 with egg yolk as substrate. The NH2-terminal sequence of the enzyme and some protein fragments obtained from its tryptic digestion were also determined. All sequences obtained were similar to those of sPLA2-IIA. The enzyme also showed good stability in the presence of organic solvents, acidic and alkaline pH media and high temperature conditions. Thus, the purified enzyme exhibited a number of unique and promising properties, making it a potential possible candidate for future applications in the treatment of phospholipid-rich industrial effluents and synthesis of useful preparations for the food production and processing industry. </span