Results of investigation of phospholipase activity and virulence of pathogenic leptospirae on a solid nutrient medium using the method of agar layers with 1% of L-lecithin in the medium of the second layer are presented. It has been demonstrated that only one zone of translucent medium is formed around the colonies of pathological leptospirae, which is obviously due to the release of phospholipase A. The examined virulent strains of pathogenic leptospirae were found to exhibit greater phospholipase activity (width of the translucent zones being 5.0 +/- 0.34 mm) than the avirulent strains (width of the zones being 1.5 +/- 0.11 mm).

• MeSH
• agar MeSH
• fosfatidylcholiny MeSH
• fosfolipasy A metabolismus   MeSH
• fosfolipasy metabolismus   MeSH
• kultivační média MeSH
• Leptospira enzymologie   patogenita   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• agar MeSH
• fosfatidylcholiny MeSH
• fosfolipasy A MeSH
• fosfolipasy MeSH
• kultivační média MeSH

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

• Klíčová slova
• antitumour activity, catalytic zinc cluster, glycoproteins, oligomerization, phospholipase C-like activity, plant nucleases,
• MeSH
• deoxyribonukleasy chemie   metabolismus   MeSH
• fosfolipasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• multienzymové komplexy chemie   metabolismus   MeSH
• myši MeSH
• rostlinné proteiny chemie   metabolismus   MeSH
• Solanum lycopersicum enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleasy MeSH
• fosfolipasy MeSH
• multienzymové komplexy MeSH
• rostlinné proteiny MeSH

Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC activity, to phosphatidic acid (PA) that participates in ABA signaling pathways. Here we describe a step-by-step protocol, which can be used to determine in situ or in vitro NPC activity. Determination is based on quantification of fluorescently labeled DAG as a product of cleavage of the fluorescently labeled substrate lipid, phosphatidylcholine. High-performance thin-layer chromatography is used for separation of fluorescent DAG. The spot is visualized with a laser scanner and the relative amounts of fluorescent DAG are quantified using imaging software.

• MeSH
• chromatografie na tenké vrstvě MeSH
• diglyceridy metabolismus   MeSH
• enzymatické testy metody   MeSH
• fluorescenční barviva metabolismus   MeSH
• fosfolipasy typu C metabolismus   MeSH
• referenční standardy MeSH
• rostliny enzymologie   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-diacylglycerol MeSH Prohlížeč
• diglyceridy MeSH
• fluorescenční barviva MeSH
• fosfolipasy typu C MeSH

• MeSH
• adenylátcyklasový toxin * MeSH
• adenylátcyklasy MeSH
• aktivace enzymů MeSH
• cholerový toxin MeSH
• faktory virulence rodu Bordetella * MeSH
• fosfolipasy MeSH
• pertusový toxin MeSH
• Publikační typ
• dopisy MeSH
• komentáře MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• adenylátcyklasy MeSH
• cholerový toxin MeSH
• faktory virulence rodu Bordetella * MeSH
• fosfolipasy MeSH
• pertusový toxin MeSH

The effect of various physico-chemical factors on production of intra- and extracellular phospholipase A1 by Salmonella newport was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth, after 12 h of incubation at 37 degrees C. Highest level of extracellular phospholipase A1, however, was seen in synthetic medium (pH 7.0) after 24 h of incubation at 37 degrees C. Agitation during incubation had no effect on the intracellular enzyme synthesis but enhanced extracellular enzyme levels. Addition of surfactants to the growth media significantly decreased both intra- and extracellular phospholipase A1 production.

• MeSH
• aerobióza MeSH
• fosfolipasy A biosyntéza   MeSH
• fosfolipasy A1 MeSH
• fosfolipasy biosyntéza   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média MeSH
• povrchově aktivní látky farmakologie   MeSH
• Salmonella enzymologie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasy A MeSH
• fosfolipasy A1 MeSH
• fosfolipasy MeSH
• kultivační média MeSH
• povrchově aktivní látky MeSH

OBJECTIVES: This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease. BACKGROUND: Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy. METHODS: We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable. RESULTS: PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE. CONCLUSIONS: Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.

• Klíčová slova
• ACS, CI, LDL-C, MI, MVE, Mendelian randomization, OR, RCT, SNP, acute coronary syndrome(s), cardiovascular diseases, confidence interval, drug development, epidemiology, genetics, low-density lipoprotein cholesterol, major vascular events, myocardial infarction, odds ratio, randomized clinical trial, sPLA(2), secretory phospholipase A(2), single-nucleotide polymorphism,
• MeSH
• alely MeSH
• celosvětové zdraví MeSH
• DNA genetika   MeSH
• incidence MeSH
• kardiovaskulární nemoci enzymologie   epidemiologie   genetika   MeSH
• lidé MeSH
• mendelovská randomizace metody   MeSH
• regulace genové exprese * MeSH
• sekreční fosfolipasy A2 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• Názvy látek
• DNA MeSH
• sekreční fosfolipasy A2 MeSH

As phospholipases of mycoplasma species may play a role in the pathogenesis of respiratory tract and urogenital tract diseases Mycoplasma mycoides and Acholeplasma laidlawii were examined as to the production of phospholipase A2 (PLA) and attempts were made to purify and characterize it. Both species produced PLA. The purified enzyme was found to be heat-labile, active at alkaline pH, revealing a single band in polyacrylamide gel electrophoresis. Metal ions such as calcium and barium, increased its activity whereas solvents at high concentrations decreased it. It was resistant to surfactants.

• MeSH
• Acholeplasma laidlawii enzymologie   MeSH
• extracelulární prostor enzymologie   MeSH
• fosfolipasy A izolace a purifikace   metabolismus   MeSH
• fosfolipasy A2 MeSH
• fosfolipasy metabolismus   MeSH
• gelová chromatografie MeSH
• koncentrace vodíkových iontů MeSH
• Mycoplasma mycoides enzymologie   MeSH
• vápník farmakologie   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasy A MeSH
• fosfolipasy A2 MeSH
• fosfolipasy MeSH
• vápník MeSH

Gangliosides released from tumor cells, as well as administered exogenously, suppress the immune responses by largely unknown mechanisms. We show here that a pretreatment of rat basophilic leukemia cells with isolated brain gangliosides inhibited the release of preformed secretory mediators from cells activated via FcepsilonRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also affected the cell-substrate adhesion and the levels of polymeric filamentous actin in Ag-activated cells. Although the production of phosphoinositides was also decreased, enzymatic activity of phosphatidylinositol 3-kinase was not inhibited. Gangliosides had no or only marginal effect on the association of aggregated FcepsilonRI with glycosphingolipid-enriched membranes and on tyrosine phosphorylation of FcepsilonRI and the linker for activation of T cells. Though pretreatment with gangliosides did not inhibit the association of linker for activation of T cells with phospholipase C (PLC)gamma1 and PLCgamma2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca(2+) mobilization. The combined data support the concept that exogenously administered gangliosides interfere with those properties of glycosphingolipid-enriched membranes that are important for the formation of plasma membrane-associated signaling assemblies containing PLCgamma but not for initial tyrosine phosphorylation of FcepsilonRI subunits.

• MeSH
• aktivace enzymů účinky léků   imunologie   MeSH
• degranulace buněk účinky léků   imunologie   MeSH
• fosfolipasa C gama MeSH
• fosfolipasy typu C antagonisté a inhibitory   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• gangliosidy farmakologie   MeSH
• glykosfingolipidy metabolismus   MeSH
• imunosupresiva farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mastocyty účinky léků   enzymologie   imunologie   metabolismus   MeSH
• membránové mikrodomény účinky léků   metabolismus   MeSH
• nádorové buněčné linie MeSH
• receptory IgE antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• signální transdukce účinky léků   imunologie   MeSH
• tyrosin antagonisté a inhibitory   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfolipasa C gama MeSH
• fosfolipasy typu C MeSH
• gangliosidy MeSH
• glykosfingolipidy MeSH
• imunosupresiva MeSH
• inhibitory enzymů MeSH
• receptory IgE MeSH
• tyrosin MeSH

Mitochondrial uncoupling protein-2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. Here we tested the hypothesis that UCP2 provides feedback downregulation of oxidative stress in vivo via synergy with an H2O2-activated mitochondrial calcium-independent phospholipase A2 (mt-iPLA2). Tert-butylhydroperoxide or H2O2 induced free fatty acid release from mitochondrial membranes as detected by gas chromatography/mass spectrometry, which was inhibited by r-bromoenol lactone (r-BEL) but not by its stereoisomer s-BEL, suggesting participation of mt-iPLA2γ isoform. Tert-butylhydroperoxide or H2O2 also induced increase in respiration and decrease in mitochondrial membrane potential in lung and spleen mitochondria from control but not UCP2-knockout mice. These data suggest that mt-iPLA2γ-dependent release of free fatty acids promotes UCP2-dependent uncoupling. Upon such uncoupling, mitochondrial superoxide formation decreased instantly also in the s-BEL presence, but not when mt-iPLA2 was blocked by R-BEL and not in mitochondria from UCP2-knockout mice. Mt-iPLA2γ was alternatively activated by H2O2 produced probably in conjunction with the electron-transferring flavoprotein:ubiquinone oxidoreductase (ETFQOR), acting in fatty acid β-oxidation. Palmitoyl-d,l-carnitine addition to mouse lung mitochondria, respiring with succinate plus rotenone, caused a respiration increase that was sensitive to r-BEL and insensitive to s-BEL. We thus demonstrate for the first time that UCP2, functional due to fatty acids released by redox-activated mt-iPLA2γ, suppresses mitochondrial superoxide production by its uncoupling action. In conclusion, H2O2-activated mt-iPLA2γ and UCP2 act in concert to protect against oxidative stress.

• MeSH
• antioxidancia metabolismus   MeSH
• buněčné dýchání účinky léků   MeSH
• down regulace účinky léků   MeSH
• fosfolipasy A2, skupina VI metabolismus   MeSH
• iontové kanály metabolismus   MeSH
• játra cytologie   MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie účinky léků   enzymologie   metabolismus   MeSH
• myši MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• peroxid vodíku farmakologie   MeSH
• slezina cytologie   MeSH
• superoxidy metabolismus   MeSH
• terc-butylhydroperoxid farmakologie   MeSH
• uncoupling protein 2 MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• fosfolipasy A2, skupina VI MeSH
• iontové kanály MeSH
• mastné kyseliny MeSH
• mitochondriální proteiny MeSH
• peroxid vodíku MeSH
• Pla2g6 protein, mouse MeSH Prohlížeč
• superoxidy MeSH
• terc-butylhydroperoxid MeSH
• Ucp2 protein, mouse MeSH Prohlížeč
• uncoupling protein 2 MeSH

A chemiluminescent flow-sensing device for the determination of phospholipase D (PLD) activity and/or choline (Ch) in biological samples using choline oxidase (ChO) and horseradish peroxidase (HRP) immobilized on Eupergit C (polymer beads of methacrylamide, N-methylene-bis-methacrylamide, and allyl-glycidyl-ether) was developed. The best results were obtained with immobilized ChO and HRP at a polymer beads wet weight ratio of 16:1. The optimized parameters of the developed sensing device were 56 microM luminol in working solution; sample volume, 60 microliters; flow rate, 0.3 ml/min; and sample throughput, 15/h. The detection limit (3 SD) using a luminescent enhancer was 1.2 microM for Ch, corresponding to 0.167 mIU of PLD activity per milliliter. Without enhancer the values were 3.0 microM and 0.417 mIU, respectively. The Ch recovery varied between 80.4 and 109%. The biological samples quenched the luminescent light to different extents, and this matrix effect was readily overcome by measuring the luminescent signal of added Ch standard. The flow biosensor was used for the determination of PLD in samples of different origin, including rape seeds during maturation.

• MeSH
• alkoholoxidoreduktasy chemie   metabolismus   MeSH
• biochemie přístrojové vybavení   MeSH
• biosenzitivní techniky MeSH
• cholin analýza   MeSH
• fosfolipasa D analýza   metabolismus   MeSH
• játra enzymologie   MeSH
• křenová peroxidasa chemie   metabolismus   MeSH
• luminiscenční měření * MeSH
• rostliny enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• alkoholoxidoreduktasy MeSH
• cholin MeSH
• choline oxidase MeSH Prohlížeč
• fosfolipasa D MeSH
• křenová peroxidasa MeSH