The deacylation of phosphatidylinositol by rat brain preparations

1979 ◽  
Vol 57 (12) ◽  
pp. 1359-1367 ◽  
Author(s):  
T. Y. P. Shum ◽  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Rat Brain ◽  
Divalent Cations ◽  
Brain Homogenate ◽  
Phospholipase A ◽  
Ph Optimum ◽  
Major Activity ◽  
Nonionic Detergent

The deacylation of phosphatidylinositol (PI) in rat brain was studied in vitro. Using 1-acyl, 2-[1-14C-oleoyl] sn-glycerol-3-phosphoinositol and [U-14C]phosphatidylinositol as substrates, a release of 14C-free fatty acid was found when incubations were conducted with the PI labelled in the 2 position, both in the 12 000 – 106 000 g pellet (microsomal) and in the 106 000 g supernate prepared from rat brain homogenate. With the 106 000 g pellet the deacylation activity was linear with time up to 15 min and was directly proportional to the amount of protein added. Two pH optima were observed, one in the region of pH 7.5 which constituted the major activity and the other in the region of pH 6.0. The apparent Km for the enzyme activity at pH 7.5 was found to be 6.2 × 10−4 M and the Vmax was 1.24 nmol of [14C]oleic acid released per minute per milligram of protein. The Ca2+ ion stimulated the activity maximally at 5 mM while other divalent cations such as Mg2+, Mn2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, Pb2+, and Zn2+ either partially or completely inhibited the activity. The nonionic detergent, Triton X-100, stimulated the deacylation more than twofold at a concentration of 0.01%. The sulfydryl reagents, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide showed partial inhibition of the reaction which was reversed by the addition of dithiothreitol. The deacylation activity in the 106 000 g supernate from rat brain was found to be directly proportional to the amount of protein added, and to the time (up to 15 min). A pH optimum was observed in the region of pH 6.0. Substrate concentration studies showed that the apparent Km was 5.0 × 10−4 M and the Vmax was 3.93 nmol of [14C]oleic acid released per minute per milligram of protein. "lyso-PI," diacylglycerol, and fatty acid were formed at pH 6.0 and pH 7.5. The data obtained indicate that from 54 (pH 7.5) to 70% (pH 6.0) of the altered PI is due to phospholipase A2 activity, 24 (pH 6.0) to 28% (pH 7.5) is due to phospholipase "C-like" activity, and from 2 (pH 6.0) to 22% (pH 7.5) may be due to phospholipase A1 activity. These results provide evidence for the deacylation component of a deacylation–reacylation cycle for the generation of specific molecular species of PI.

Metabolism of phosphoglycerides in E. coli IV. The positional specificity and properties of phospbolipase A

1969 ◽  
Vol 47 (12) ◽  
pp. 1125-1128 ◽  
Author(s):  
P. Proulx ◽  
C. K. Fung
Keyword(s):  
Sodium Lauryl Sulfate ◽  
Divalent Cations ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Phospholipase A ◽  
Lauryl Sulfate ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
E Coli

The hydrolysis of labelled phosphatidylethanolamine by E. coli was studied in vitro. Phospholipase A, as detected by 32P-labelled lysophosphatidylethanolamine formation, had two pH optima, 5 and 8.4. On the other hand lysophosphofipase was active only in the alkaline range, had a pH optimum of 10, and was inhibited by high concentrations of either sodium deoxycholate or sodium lauryl sulfate. Phospholipase A required Ca2+ addition for maximal activity at both pH optima. Mg2+ also stimulated the activity but other divalent cations tested were slightly inhibitory or without effect. Sodium lauryl sulfate completely inhibited at pH 5. Experiments with singly and doubly labelled phosphatidylethanolamine indicated that phospholipase A1 activity was predominant at both acid and alkaline pH. Lower levels of phospholipase A2 were detectable only at alkaline pH.

In vitro anticholinesterase, antimonoamine oxidase and antioxidant properties of alkaloid extracts from kola nuts (Cola acuminata and Cola nitida)

2018 ◽  
Vol 16 (1) ◽  
Author(s):  
Ganiyu Oboh ◽  
Ayokunle O. Ademosun ◽  
Opeyemi B. Ogunsuyi ◽  
Esther T. Oyedola ◽  
Tosin A. Olasehinde ◽  
...  
Keyword(s):  
Side Effects ◽  
Neurodegenerative Diseases ◽  
Rat Brain ◽  
Brain Homogenate ◽  
Oh Radicals ◽  
Mao Inhibitors ◽  
Alkaloid Extracts ◽  
Cola Nitida ◽  
Cola Acuminata

Abstract Background The development of cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for management of neurodegenerative diseases such as Alzheimer’s disease (AD) has come with their undesirable side effects. Hence, research for potent but natural ChE and MAO inhibitors with little or no side effects is essential. This study investigated the potentials of alkaloid extracts from two Cola species as nutraceuticals for prevention and management of AD. Methods Alkaloid extracts were obtained from two Cola species (Cola nitida [KN] and Cola acuminata [KA]) by solvent extraction method. The extracts were characterized for their alkaloid contents using gas chromatography (GC). The effects of the extracts on ChE and MAO activities were investigated in vitro. Also, the extracts’ ability to inhibit Fe2+-induced lipid peroxidation in rat brain homogenate, scavenge DPPH and OH radicals, as well as chelate Fe2+ were determined. Results GC characterization revealed the presence of augustamine and undulatine as the predominant alkaloids in the extracts. There was no significant (P > 0.05) difference in the inhibitory effects of the extracts on ChE activities. However, KA extract exhibited significantly higher (P < 0.05) MAO inhibitory effect than KN. Also, KA extract inhibited Fe2+- induced malondialdehyde (MDA) production in rat brain homogenate more significantly than KN, while there was no significant difference in DPPH and OH radicals scavenging, as well as Fe2+-chelating abilities of the extracts. Conclusions Our findings revealed that KN and KA alkaloid extracts exhibited significant effect in vitro on biological pathways that may contribute to neuroprotection for the management of neurodegenerative diseases.

Neoplastic and pharmacological influence on the permeability of an in vitro blood-brain barrier

1995 ◽  
Vol 82 (6) ◽  
pp. 1053-1058 ◽  
Author(s):  
Paul A. Grabb ◽  
Mark R. Gilbert
Keyword(s):  
Endothelial Cell ◽  
Rat Brain ◽  
Phospholipase A ◽  
Cell Permeability ◽  
Capillary Endothelium ◽  
Brain Capillary ◽  
Content Type ◽  
Endothelial Cell Permeability ◽  
Fine Print

✓ The authors investigated the effects of glioma cells and pharmacological agents on the permeability of an in vitro blood-brain barrier (BBB) to determine the following: 1) whether malignant glia increase endothelial cell permeability; 2) how glucocorticoids affect endothelial cell permeability in the presence and absence of malignant glia; and 3) whether inhibiting phospholipase A2, the enzyme that releases arachidonic acid from membrane phospholipids, would reduce any malignant glioma—induced increase in endothelial cell permeability. Primary cultures of rat brain capillary endothelium were grown on porous membranes; below the membrane, C6, 9L rat glioma, T98G human glioblastoma, or no cells (control) were cocultured. Dexamethasone (0.1 µM), bromophenacyl bromide (1.0 µM), a phospholipase A2 inhibitor, or nothing was added to culture media 72 hours prior to assaying the rat brain capillary endothelium permeability. Permeability was measured as the flux of radiolabeled sucrose across the rat brain capillary endothelium monolayer and then calculated as an effective permeability coefficient (Pe). When neither dexamethasone nor bromophenacyl bromide was present, C6 cells reduced the Pe significantly (p < 0.05), whereas 9L and T98G cells increased Pe significantly (p < 0.05) relative to rat brain capillary endothelium only (control). Dexamethasone reduced Pe significantly for all cell preparations (p < 0.05). The 9L and T98G cell preparations coincubated with dexamethasone had the lowest Pe of all cell preparations. The Pe was not affected in any cell preparation by coincubation with bromophenacyl bromide (p > 0.45). These in vitro BBB experiments showed that: 1) malignant glia, such as 9L and T98G cells, increase Pe whereas C6 cells probably provide an astrocytic influence by reducing Pe; 2) dexamethasone provided significant BBB “tightening” effects both in the presence and absence of glioma cells; 3) the in vivo BBB is actively made more permeable by malignant glia and not simply because of a lack of astrocytic induction; 4) tumor or endothelial phospholipase A2 activity is probably not responsible for glioma-induced increased in BBB permeability; and 5) this model is useful for testing potential agents for BBB protection and for studying the pathophysiology of tumor-induced BBB disruption.

scholarly journals Fatty acid is a potential agent for bone tissue induction: In vitro and in vivo approach

2017 ◽  
Vol 242 (18) ◽  
pp. 1765-1771 ◽  
Author(s):  
Guinea BC Cardoso ◽  
Erivelto Chacon ◽  
Priscila GL Chacon ◽  
Pedro Bordeaux-Rego ◽  
Adriana SS Duarte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Bone Tissue ◽  
In Vivo Studies ◽  
Surrounding Tissue ◽  
Bone Induction ◽  
Acid Addition

Our hypothesis was to investigate the fatty acid potential as a bone induction factor. In vitro and in vivo studies were performed to evaluate this approach. Oleic acid was used in a 0.5 wt.% concentration. Polycaprolactone was used as the polymeric matrix by combining solvent-casting and particulate-leaching techniques, with a final porosity of 70 wt.%, investigated by SEM images. Contact angle measurements were produced to investigate the influence of oleic acid on polycaprolactone chains. Cell culture was performed using adipocyte-derived stem cells to evaluate biocompatibility and bioactivity properties. In addition, in vivo studies were performed to evaluate the induction potential of oleic acid addition. Adipocyte-derived stem cells were used to provide differentiation after 21 days of culture. Likewise, information were obtained with in vivo data and cellular invagination was observed on both scaffolds (polycaprolactone and polycaprolactone /oleic acid); interestingly, the scaffold with oleic acid addition demonstrated that cellular migrations are not related to the surrounding tissue, indicating bioactive potential. Our hypothesis is that fatty acid may be used as a potential induction factor for bone tissue engineering. The study’s findings indicate oleic acid as a possible agent for bone induction, according to data on cell differentiation, proliferation, and migration. Impact statement The biomaterial combined in this study on bone regeneration is innovative and shows promising results in the treatment of bone lesions. Polycaprolactone (PCL) and oleic acid have been studied separately. In this research, we combined biomaterials to assess the stimulus and the speed of bone healing.

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

STUDIES ON THE ACYLATION OF LYSOLECITHINS

1963 ◽  
Vol 41 (9) ◽  
pp. 1983-1990 ◽  
Author(s):  
N. H. Tattrie ◽  
J. R. Bennett
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Sodium Methoxide ◽  
Cadmium Chloride ◽  
Phospholipase A ◽  
Chloride Complexes ◽  
Egg Lecithin ◽  
Hydrolysis Of

"Semi-synthetic" lecithins were prepared by the acylation of lysolecithin cadmium chloride complexes (the lysolecithins were produced from egg lecithin by hydrolysis with phospholipase A) in the presence of pyridine with 1-C14 myristoyl chloride. The labelled lecithins were hydrolyzed with phospholipase A to yield free fatty acids (94% of the label) and lysolecithins (6% of the label). However, when lysolecithins were enzymically acylated with 1-C14 linoleic acid and the resulting labelled lecithins cleaved with phospholipase A, no labelled fatty acid was detected in the lysolecithin fraction.Labelled lecithins were also enzymically synthesized with 1-C14 linoleic acid and lysolecithins prepared from egg lecithin by reaction with sodium methoxide. The labelled lecithins were hydrolyzed with phospholipase A and the labelled lysolecithins were isolated and reacylated enzymically with oleic acid. The hydrolysis of these lecithins with phospholipase A showed that the lysolecithins retained all the label.

scholarly journals Antioxidative Activity of Ferrocenes Bearing 2,6-Di-Tert-Butylphenol Moieties

2010 ◽  
Vol 2010 ◽  
pp. 1-6 ◽  
Author(s):  
E. R. Milaeva ◽  
S. I. Filimonova ◽  
N. N. Meleshonkova ◽  
L. G. Dubova ◽  
E. F. Shevtsova ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Rat Brain ◽  
Antioxidative Activity ◽  
Neuroprotective Effect ◽  
In Vitro Study ◽  
Brain Homogenate ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Calcium Dependent

The antioxidative activity of ferrocenes bearing either 2,6-di-tert-butylphenol or phenyl groups has been compared using DPPH (1,1-diphenyl-2-picrylhydrazyl) test and in the study of the in vitro impact on lipid peroxidation in rat brain homogenate and on some characteristics of rat liver mitochondria. The results of DPPH test at20∘C show that the activity depends strongly upon the presence of phenolic group but is improved by the influence of ferrocenyl fragment. The activity of N-(3,5-di-tert-butyl-4-hydroxyphenyl)iminomethylferrocene (1), for instance, was 88.4%, which was higher than the activity of a known antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) (48.5%), whereas the activity of N-phenyl-iminomethylferrocene2was almost negligible−2.9%. The data obtained demonstrate that the compounds with 2,6-di-tert-butylphenol moiety are significantly more active than the corresponding phenyl analogues in the in vitro study of lipid peroxidation in rat brain homogenate. Ferrocene1performs a promising behavior as an antioxidant and inhibits the calcium-dependent swelling of mitochondria. These results allow us to propose the potential cytoprotective (neuroprotective) effect of ditopic compounds containing antioxidant 2,6-di-tert-butylphenol group and redox active ferrocene fragment.

scholarly journals Fatty Acid Composition, Antioxidant, and in vitro Anti-inflammatory Activity of Five Cold-Pressed Prunus Seed Oils, and Their Anti-biofilm Effect Against Pathogenic Bacteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Florinda Fratianni ◽  
Antonio d'Acierno ◽  
Maria Neve Ombra ◽  
Giuseppe Amato ◽  
Vincenzo De Feo ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Inflammatory Activity ◽  
Seed Oils ◽  
Black Cherry ◽  
Dpph Assay ◽  
Anti Inflammatory ◽  
Cold Pressed

Background/Aim: Sweet almond (Prunus amygdalus dulcis) oil is one of the most famous cold-pressed seed oils. However, other species of Prunus can provide oils with healthy properties. We analyzed the fatty acid (FA) composition, as well as the antioxidant, the in vitro anti-inflammatory properties, and the antibiofilm activity of five commercial vegetable cold-pressed seed oils of apricot, peach, plum, cherry, and black cherry.Methods: Gas Chromatography-Mass Spectrometry was performed for the analysis of FAs The antioxidant property of the oils was carried using different tests [2, 2-diphenyl-1-picrylhydrazyl (DPPH assay)], Ferric Reducing Antioxidant Power (FRAP), and the 2, 20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+). The denaturation assay performed on bovine serum albumin (BSA) was used to evaluate the in vitro anti-inflammatory activity. The anti-biofilm activity was assessed using five pathogenic strains, namely, Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, through the crystal violet test and the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT), used to evaluate the metabolism of the microbial cells present within the biofilm.Results: Oleic acid and linoleic acids were the most abundant FAs. Black cherry seed oil exhibited the best antioxidant activity, but in general, the amount of oil needed to inhibit the activity of 1 ml of DPPH assay at 50% did not exceed 10 μg. The extract concentration for the 50% inhibition of the denaturation of the protein (IC50) did not exceed 4.4 μg. Linoleic and stearic acids affected the antioxidant activity of the oils; oleic acid, linolenic, and palmitoleic acids exhibited beneficial effects in preserving the BSA denaturation, as shown by the correlation data. The oils were able to inhibit the biofilm formation of the pathogens (up to 71.40% of inhibition) as well as act against their mature biofilm, although with different strengths, with values up to 61.54%. Concurrently, they also acted on the pathogen metabolism.Conclusion: The oils represent a valuable source of some healthy FAs. They showed potential antioxidant and anti-inflammatory in vitro activity, in addition, their potential effect on the biofilm can offer important ideas for research and reflection on their use as functional foods and/or ingredients.

Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions

1976 ◽  
Vol 54 (3) ◽  
pp. 249-260 ◽  
Author(s):  
H. H. Bishop ◽  
K. P. Strickland
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Rat Brain ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Distribution ◽  
Assay Method ◽  
Detergent Solution ◽  
Significant Activity ◽  
Ph Optimum ◽  
Phosphatidic Acids

The enzyme, CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer–detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleoylphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60 mM for Mg2+, 6 mM for CTP and 250 μg per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90 000 × g fraction (microsomal) with small but significant activity in the 12 000 × g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses): 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl- (3.85), 1-stearoyl-2-arachidonoyl- (3.31), 1-arachidonoyl-2-stearoyl- (3.16), 1,2-diarachidonoyl- (0.72), 1,2-dicaproyl- (0.67), 1,2-dipalmitoyl- (0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.

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