Detection Of Phospholipase Research Articles

Detection of Phospholipase and Asparty1 Protaines gens in Candida Species Isolated From Mammary Candidiasis in Lactating Women

Breast and nipple thrush (BNT) is a yeast infection of the nipple and breast caused by a fungal organism knownas Candida albicans, a common cause of all thrush infections. It occurs most commonly in breastfeedingmothers. The infection may lead to severe nipple and breast pain, especially during breastfeeding. Thecurrent study was conducted at Al-Kut hospital for Gynecology, obstetrics and pediatrics, and TaibaSpecialist Centerin Wasit province. (162) milk samples were collected from lactating wommen suspected tohave mammary candidiasis infections according to the clinical manifestations diagnosed by the examiningphysician and patient with symptom , then, the samples were examined by optical microscopy and celldetection which show evidence of the presence of pathogenic yeast. Samples were cultured in sabouraudagar and then in chrom agar to classify the organisms, and they were held for further conformation and todetect the most common virulence factor gen by polymerase chain reaction (PCR) Molecular identificationof Candida spp in this study, done by conventional PCR by partial amplification gene encoding for largesubunit of 18S rRNA gene by specific primer sequences. The yield of the detection C. albicans and C.parapsilosis, was 37/37(100%) (100%) and 15/15 (100%), respectively. The results of PCR amplificationof( SAP1-8)genes in Candida isolates (61) were SAP1 gen was present in 61/61(100%) , SAP2 gen was54/61(96%), SAP3 gen was 45/61(86%), SAP4 gen was 35/61(76%), SAP5 gen was 32/61(66%), SAP6 genwas 45/61(60%), SAP7 gen was 35/61(30%) and SAP8 gen was 21/61(34%). By molecular technique usingPCR (PLB1-2 gene) and sequencing, it was seen that Seventeen isolates belong C. albicans were positivefor PLB1 gene 17/61 (91.6%). Fourteen isolates belong C. albicans were positive for PLB1 gene 14/61.

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions. We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

Detection of phospholipase D on solid materials

Detection of phospholipase D on solid materials

Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?

Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.

Molecular weight variations in the diversity of phospholipase A 2 forms in reptile venoms

Reptile venoms exhibit a wide diversity of phospholipase A 2 forms when examined by electrophoretic and chromatographic techniques which separate on the basis of net charge. In principle, diversity in charge among the enzyme forms could result from two type of structural modifications: (i) pretranslational modifications, such as differences in amino acid sequences; (ii) post-translational modifications, such as partial proteolysis or hydrolysis of amide functions of asparagine and glutamine residues. Some types of modifications alter both charge and molecular weigth. A radiolabeling technique has been developed which allows detection of phospholipase A 2 enzymes after electrophoretic separation on the basis of molecular weight in polyacrylamide gels containing sodium dodecylsulfate. Venom preparations were radiolabeled by incubation with p- dodecylphenacylbromide , which was prepared radiolabeled to high specific activity with tritium. The labeling agent was dispersed in phosphatidylcholine liposomes and incubated with venom preparations under conditions of optimal enzyme activity. The preparations were then extracted with ether and hexane to remove substrate and unbound label and subjected to polyacrylamide gel electrophoresis under standard conditions. Protein-bound radiolabel was detected by fluorography and autoradiography. Patterns of labeled species obtained by gel electrophoresis of selected venoms using separation on the basis of charge were similar to the patterns of phospholipase A 2 variants obtained under the same electrophoretic conditions using enzymatic detection in phosphatidylcholine-containing gels. Examination of a variety of snake and lizard venoms using this technique revealed the presence of multiple molecular weight forms of labeled enzymes, but the extent of diversity was less than that observed with the same venoms using separation of phospholipase electrophoretic variants on the basis of charge. The results are consistent with diversity in reptile venom phospholipase A 2 forms being derived from differences in both molecular weight and charge.

Studies on enzymes related to diacylglycerol production in activated platelets: I. phosphatidylinositol-specific phospholipasec: Further characterization using a simple method for determination of activity

A simple method of determination of phosphatidylinositol-specific phospholipase C activity in soluble platelet extracts has been devised. It is based on the use of a total lipid extract from rat liver microsomes incubated with [ 3H]inositol in the presence of MnCl 2. Phosphatidylinositol hydrolysis can thus be detected by determining hydrosoluble radioactivity formed upon incubation with enzyme fractions. Owing to the presence of other phospholipids in the assay system, phospholipase C was inhibited. However, activity was restored by sodium deoxycholate (0.1%, w/v). Optimal conditions also included calcium (1–10 mM) and a pH between 5 and 7, allowing the detection of phospholipase C without the need for purifying the substrate. Using this simplified procedure, platelet phospholipase C was submitted to preparative electrofocusing and to gel filtration chromatography on Sephacryl S-200. Phospholipase C focused in one single peak at pH 6.1. An M r of 86000 was found upon gel chromatography of a crude extract, against 68 000 when phospholipase C had been previously purified by electrofocusing. These data indicate that phospholipase C might be associated with lipids or with an M r 20 000 protein, the significance of which is discussed.

Detection of phospholipase C and hemolysin in the cultures of several Streptoverticillium.

Detection of phospholipase C and hemolysin in the cultures of several Streptoverticillium.