scholarly journals Validation of a simple radiochemical assay measuring hydrolysis of choline-labelled microsomal phosphatidylcholine by phospholipase C. pH-dependence

1976 ◽  
Vol 160 (3) ◽  
pp. 475-479 ◽  
Author(s):  
B R Cater ◽  
P Trivedi ◽  
T Hallinan
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Ph Dependence ◽  
Selective Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens. Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0.

Survival of Bacillus cereus Vegetative Cells and Spores during In Vitro Simulation of Gastric Passage

2012 ◽  
Vol 75 (4) ◽  
pp. 690-694 ◽  
Author(s):  
SIELE CEUPPENS ◽  
MIEKE UYTTENDAELE ◽  
KATRIEN DRIESKENS ◽  
ANDREJA RAJKOVIC ◽  
NICO BOON ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Acid Resistance ◽  
In Vitro Experiments ◽  
Vegetative Cells ◽  
Ph Values ◽  
Constant Ph ◽  
In Vitro Simulation ◽  
Ph Range

The enteric pathogen Bacillus cereus must survive gastric passage in order to cause diarrhea by enterotoxin production in the small intestine. The acid resistance and the survival after gastric passage were assessed by in vitro experiments with acidified growth medium and gastric simulation medium with B. cereus NVH 1230-88 vegetative cells and spores. First, batch incubations at constant pH values for 4 h, which represented different physiological states of the stomach, showed that spores were resistant to any gastric condition in the pH range of 2.0 to 5.0, while vegetative cells were rapidly inactivated at pH values of ≤4.0. Second, a dynamic in vitro gastric experiment was conducted that simulated the continuously changing in vivo conditions due to digestion dynamics by gradually decreasing the pH from 5.0 to 2.0 and fractional emptying of the stomach 30 to 180 min from the start of the experiment. All of the B. cereus spores and 14% (±9%) of the vegetative cells survived the dynamic simulation of gastric passage.

The binding and hydrolysis of sphingomyelin by phospholipase C (Bacillus cereus) as shown by 31P NMR

1984 ◽  
Vol 16 (8) ◽  
pp. 931-934 ◽  
Author(s):  
Kjf.ll M. Aalmo ◽  
Jostein Krane ◽  
Clive Little ◽  
Carlyle B. Storm
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
31P Nmr ◽  
Hydrolysis Of

scholarly journals DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 262 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Protein Docking ◽  
Target Protein ◽  
Current Work ◽  
Side Chain ◽  
Contact Points ◽  
Enzymatic Function ◽  
Target Enzyme

The ability to accurately and effectively predict the interaction between proteins and small drug-like compounds has long intrigued researchers for pedagogic, humanitarian and economic reasons. Protein docking methods (AutoDock, GOLD, DOCK, FlexX and Glide to name a few) rank a large number of possible conformations of protein-ligand complexes using fast algorithms. Previously, it has been shown that structural congruence leading to the same enzymatic function necessitates the congruence of electrostatic properties (CLASP). The current work presents a methodology for docking a ligand into a target protein, provided that there is at least one known holoenzyme with ligand bound - DOCLASP (Docking using CLASP). The contact points of the ligand in the holoenzyme defines a motif, which is used to query the target enzyme using CLASP. If there are no significant matches, the ligand cannot be docked in the protein. Otherwise, the holoenzyme and the target protein are superimposed based on congruent atoms. The same linear and rotational transformations are also applied to the ligand, thus creating a unified coordinate framework having the holoenzyme, the ligand and the target enzyme. This provides the docked ligand in the target enzyme. Previously, CLASP was used to predict and validate (in vivo) the inhibition of phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus by two dipeptidyl peptidase-IV (DPP4) inhibitors - vildagliptin and K-579. In the current work, vildagliptin was docked to the PI-PLC structure complexed with myo-inositol using DOCLASP. The docked ligand is free from steric clashes and interacts with the same side chain residues that bind myo-inositol, providing corroboration of the validity of the proposed methodology.

Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum.

1986 ◽  
Vol 32 (8) ◽  
pp. 1503-1505 ◽  
Author(s):  
E Sykes ◽  
F L Kiechle ◽  
E Epstein
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Cereus ◽  
Molecular Mass ◽  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Alkaline Phosphatase Activity ◽  
Plasma Membranes ◽  
High Molecular Mass ◽  
Serum Samples ◽  
Liver Membrane

Abstract Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin. This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes. Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band. Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.

scholarly journals NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

1994 ◽  
Vol 14 (1) ◽  
pp. 646-654 ◽  
Author(s):  
T Johansen ◽  
G Bjørkøy ◽  
A Overvatn ◽  
M T Diaz-Meco ◽  
T Traavik ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Constitutive Expression ◽  
Soft Agar ◽  
3T3 Cells ◽  
Gene Encoding ◽  
Stable Transfectants ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.

Kinetics of the Hydrolysis of Monodispersed and Micellar Phosphatidylcholines Catalyzed by a Phospholipase C from Bacillus cereus

1991 ◽  
Vol 110 (1) ◽  
pp. 88-95 ◽  
Author(s):  
Kiyoshi Ikeda ◽  
Seiji Inoue ◽  
Chika Amasaki ◽  
Keizo Teshima ◽  
Hiroh Ikezawa
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Kinetics Of ◽  
Hydrolysis Of

scholarly journals Evidence for Contribution of Tripartite Hemolysin BL, Phosphatidylcholine-Preferring Phospholipase C, and Collagenase to Virulence of Bacillus cereus Endophthalmitis

2000 ◽  
Vol 68 (9) ◽  
pp. 5269-5276 ◽  
Author(s):  
Douglas J. Beecher ◽  
Timothy W. Olsen ◽  
Eileen B. Somers ◽  
Amy C. L. Wong
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Bacterial Cells ◽  
Retinal Tissue ◽  
Toxic Concentrations ◽  
Hemolysin Bl

ABSTRACT Bacillus cereus causes a highly fulminant endophthalmitis which usually results in blindness. We previously concluded that hemolysin BL (HBL), a tripartite necrotizing pore-forming toxin, is a probable endophthalmitis virulence factor because it is highly toxic to retinal tissue in vitro and in vivo. We also determined that B. cereus produces additional retinal toxins that might contribute to virulence. Here we fractionated crudeB. cereus culture supernatant by anion-exchange chromatography and found that in vitro retinal toxicity was also associated with phosphatidylcholine-preferring phospholipase C (PC-PLC). The pure enzyme also caused retinal necrosis in vivo. We showed that phosphatidylinositol-specific PLC and sphingomyelinase were nontoxic and that two hemolysins, cereolysin O and a novel hemolysin designated hemolysin IV, were marginally toxic in vitro. The histopathology of experimental septic endophthalmitis in rabbits mimicked the pathology produced by pure HBL, and both HBL and PC-PLC were detected at toxic concentrations in infected vitreous fluid. Bacterial cells were first seen associated with the posterior margin of the lens and eventually were located throughout the lens cortex. Detection of collagenase in the vitreous humor suggested that infiltration was facilitated by the breakdown of the protective collagen lens capsule by that enzyme. This work supports our conclusion that HBL contributes to B. cereus virulence and implicates PC-PLC and collagenase as additional virulence factors.

scholarly journals A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes

1984 ◽  
Vol 222 (2) ◽  
pp. 535-540 ◽  
Author(s):  
B P Hughes ◽  
K A Rye ◽  
L B Pickford ◽  
G J Barritt ◽  
A H Chalmers
Keyword(s):  
Arachidonic Acid ◽  
Oleic Acid ◽  
Stearic Acid ◽  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Total Concentration ◽  
Isolated Hepatocytes ◽  
Transient Increase ◽  
Rapid Hydrolysis ◽  
Hydrolysis Of

Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes. The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min. No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon. The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+. Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols. In hepatocytes prelabelled with [14C]arachidonic acid, vasopressin increased the amount of [14C]diacylglycerol. The effects of vasopressin on the total concentration of diacylglycerols and [14C]diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens. The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.

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